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Antibody Diversity (cont.)


and the T-Cell Receptor
Mai Mazin and Sara Zuriqat
Ziad Al-Nasser
Sunday, 10/7/2011
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Immunology - Lecture 6 / Sunday, 10-07-2011
Done by: Mai Mazin & Sara Zuriqat

"This lecture covers the remainder of Ch.6 and all Ch.7"
Anti-body Diversity (Cont)
We will continue answering the question of how we are going to get around 10
11
different specificities of immunoglobulins and around 10
16
-10
18
different
specificities of the T cell receptors. We said that to answer this question we
should know the structure of the immunoglobulin.
The structure is formed by light chains and heavy chains, and those have a
variable region and a constant region. The variable region is coded by different
genes; the V genes and the J genes for the light chain, and the V, D, J for the
heavy chain. Those genes are located on different chromosomes; chromosome #2
for the kappa chain, chromosome #22 for the lambda chain, and chromosome #14
for the heavy chain.
And we said that a gene rearrangement is going to take place during B cell
development -where B cell is the one responsible for making antibodies-. When
we talk about the gene rearrangement at the germ line, we will see that the V and
the J start joining each other and we need enzymes to be responsible for this; we
call them the Recombinases or the gene rearrangement genes; RAG1 and RAG2.
Those are so important; that is if they are missing in our body then no gene
rearrangement is going to take place, and the patient is going to be severely
immune compromised. We call it "severe combined immune deficiency" if those
enzymes are missing.
When the genes are rearranged and become closer to each other, transcription is
going to take place. After the transcription process, looping out of the introns or
what we call it gene splicing is going to take place (we know we have exons and
introns in the primary RNA), and then they will come together starting with the L
(the leading sequence) and then the genes from the 5' end to the 3' end. Of
course you know that the mRNA is almost a complementary copy of the DNA.
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So the gene splicing is going to take place; the introns are going to be spliced out
while the exons come all together. Those exons go into the translation, so what
have been selected is going to be here expressed as polypeptide chain. This
polypeptide chain will be either a light chain (either kappa k or lambda )* or a
heavy chain.
*This depends on which one is going to be selected randomly of course. The same
thing is going to be applied on the heavy chain; which heavy chain is going to be
selected first.
I said previously that the and o are going to be selected first; because they are
the ones to be shown as B cell receptors. And then after the IgM & the IgD have
been developed on the surface, we have what's called excreted IgM. And when
the B cell goes into secondary immune response the same cell (memory cell) will
switch; meaning that it'll keep the variable region intact and change the heavy
chain; so instead of having IgM, we are going to have for example IgG1 (if the
switch selected the 1) with the same specificity of the chain. This is called
isotype switching.
So the antibody diversity depends on how many of those genes (the V, the J and
the D) we are having, and then the recombination that is going to take place, and
then after that gene splicing, and sometimes what we call junctional diversity that
could take place.
Junctional diversity: in the variable region the last 100 genes coded for the last
100 amino acids of the light chain. Those genes could be added or deleted at this
area and we call that junctional diversity. The enzymes needed for that called TdT
(Terminal deoxynucleotidyl transferase) that will add nucleotide into that area so
it will add to the diversity as long as it is in the variable region.
In the secondary immune response we have what's called somatic hyper-
mutation where here a mutation is going to take place to give you what we call a
better fit.
This is (slide #4) a summary of what we are going to talk about:
- 10
11
comes from the calculation of (how many V genes we have) x (how
many J genes we have) this is for the light chain x (how many V of the
heavy chain) x (how many of the D) don't know why the doctor dropped
the J genes of the heavy chain. We add into that the somatic
hypermutation and the junctional diversity and the chance of which light
chain is going to bind to which heavy chain.
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- We are dealing with heavy and light chains; variable and constant regions.
The variable region is the one responsible for the specificity while the
constant region deals with the biological function.
- Coded by different gene segments.
- Rearrangement during development; if you don't have it, you'll end up
with immune suppression. It starts with a leading sequence, j, and D genes
in addition to V and C.
- Many to select from, brought next to each other, the gene rearrangement
and the enzyme Recombinases.
- Somatic recombination, products then they assemble.
- Remember V for binding, C for biological function.

Referring to the figure; k presents on Ch.2, this is the DNA at the germ line. You
can see how the genes are arranged and one from each is going to be selected.
We have 35 variable genes of the k (the last 100 AA are coded by them). If we
select one of those V genes in addition to one of the 5J (J1-J5) plus the C region
we'll end up with the k light chain. Its specificity depends on whether we select
V1, V2, etc
*So for k light chain one of the V, J should be selected plus the C
k
..
The is the same thing, the difference is just that is on Ch.22 and here you have
more than one constant region C

1, C

2, So you have 4 different types of


and this is for biological function (has nothing to do with specificity). The one
which has to do with specificity is the variable; about 30 V and around 4 or 5 J.
In the heavy chain we have more genes so more variation; around 50 V genes and
we have about 25 diversity genes (D) and around 6 J; those they make the
variable region of the heavy chain. The D genes are the difference between the L
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& the H chains & then we have the constant region. when we are talking about it
we are talking about the isotypes and the subtypes; the classes and the
subclasses; , o, 1, 2, 3, 4, o1, o2, c. One of them is going to be selected to
make that particular constant region of the H chain.
*In the light chain; V & J
*In the heavy chain; start with D&J then the V >> VDJ

In more details of what's happening as you see here:












We start with the V, J at the germline> VJ loint by somatic recombination and DNA
rearrangement> transcription to RNA the same VJ> then the J-C intersegment in
blue is going to be spliced out> VJC> protein synthesis. Could be kappa or lambda.
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The heavy chain nearly the same; we start with the D and the J> recombination>
VDJ after the segment between V and DJ looped out> transcription from DNA into
RNA> the region between VDJ & C is going to be spliced out ending up with VDJC.
It could be for example V
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D
10
J
2
C with a certain specificity of a selection.
You can see here the carbohydrates antigen (in red) will be added in different
constant region 1, 2 and 3.
You can see the light chain has been developed. The light and heavy chains will
bind to form that particular specificity; so if you look at the colors of this particular
specificity the red for the V, the purple one for the D and yellow for the J, you can
imagine how many different options we do have. If we calculate them, they will
give up to 10
11
different specificities as I said.
Slide.9 The same point and it gives you specific example of V
3
J
2
C
K
>>

k light chain.
We have 4 different types of the constant lambda on Ch. 22 that can be expressed
in total population of Ig in an individual* and this has nothing to do with the
specificity, it has to do with the nature of the constant region.
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*however any given Ig molecule should contain 2 identical L chains.
Diversity also will be enhanced by a mutation that could be taking place at the
junctional phase between the J and the V. We call that junctional diversity where
some nucleotides will be added while some will be deleted (addition/deletion type
mutation) the enzyme required here is TdT.
So if you look here (the table below) to the initial gene segments, and what they
are going to code for, we can see that a nucleotide deletion causes the Aspargine
to be replaced by Isoleucine and this will give you an additional diversity. The
same with TdT; Glu will appear and it'll add also to the diversity.

What about the first Ig that is going to be developed? We said that IgM has to be
the first antibody to develop because it acts as the B cell receptor. We have 2
types of IgM; the surface one (presents on the surface forming the B cell
receptor) and the secreted form that is going to form the pentamer with the
same specificity, as well as the IgD plays a major role of the function of the IgM.
So both of them have to be expressed on the surface and we call that co-
expression (both the and the o heavy chains are going to be transcribed) and
then we have what we call alternative splicing; that is one of them is going to be
expressed at a time and then it will be added up to the surface so both of them
are going to be shown on the surface.
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Now look here on the B cell








How IgM & IgD are going to be added to the surface of the cell?
The most important thing is that IgM & IgD must have the same specificity and
that makes sense, and the change is going to be only in the constant region. You
can see here we have VDJ in a heavy chain of course. Then we have like a switch
region we call it polyadenylation A1 after C

. Then we have the constant delta and


we have PA2. So it depends on whether PA1 or PA2 is going to be stimulated at
the mRNA (what is going to be stimulated is going to be expressed). This is called
alternative splicing; alternative means one at a time:
Either PA1
>>
C


>>
IgM
Or PA2
>>
C
o >>
IgD
And both of them are going to be expressed on the surface, so the variable region
is the same in both & o. The difference in the C will have different functions.
What really decides for IgM to be secreted and stay on the surface of the B cell as
a B cell receptor also determined by the
gene expression.
If you look carefully, this is the surface
IgM. It has this green part of 2 parts, one
on the cytoplasmic membrane & one
inside the cell. Those (the green ones)
have to be coded by genes, while the
others as you can see they are the same.
So the difference on this
transmembrane section that's if that is
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selected (T
m
1 & T
m
2

by stimulating PA
m
) it's going to be a surface, if it's not then
it's going to be secreted.
So if you look to here; the variable region is the same> same specificity, we have
then C

, the surface part (pink) & the membranous part (green); T


m
1 & T
m
2 have
polyadenylation part. It depends on which is going to be stimulated if PA
m
is
stimulated, the green part will be added, and will determine that the resultant
protein is going to function as a part that will hold the Ig into the surface & the
IgM will act as a B cell receptor.
So B cell receptor definition: it's a monomer of IgM, the only difference between
this & the secreted form is the transmembrane part that's going to be coded by
genes, and we have the polyadenylation sites that's going to be stimulated to
code for that particular part (the green one).










This slide shows the rearrangement part & how we are going to detect that by
molecular microbiology techniques.
We get here a DNA from fibroblasts, B cancerous cells, and polyclonal cells and
you can see how those genes have been rearranged at the malignant side
compared to others that they don't fixed.
They say that these types of experimentations are nowadays old & can be
replaced with polymerase chain reaction to follow up the J, V or the D from one
location into the other.
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This is what we call allelic exclusion. Do you remember when we talked about
isotypes, allotypes, and idiotypes? We said that isotypes are the antigens that
form the heavy chain and the constant regions, they are the same within the same
species; so all of us we share the same isotypes. And when we have other
antigens that could differ we call them the allotypes, so they are added up into
the heavy chain we call them marker 1, marker 2, etc. For example, IgG1 (m
1
) if
we have marker 1.
If you have marker 1 and I inject mine into yours nothing would happen, but if you
don't have it & I do have it then you are going to respond to this marker.
Those markers are inherited in a process we call it allelic exclusion; one allele is
going to be expressed & the other will not. So if you inherit m1 & m2 only one of
them is going to be expressed (not exactly the same as MHC where we have co-
dominance; part from the mother & part from the father). So this is allelic
exclusion; one is going to be excluded & only one is going to be expressed.
So if you look above at the parents with these markers in red or blue, each one
has a marker of its own, so when you look to the inheritance of allotype1 & 2 only
one is going to be expressed on the surface either the red or the blue. No blue
and red on the same cell as those of MHC antigens. When we are talking about
allotypes we talk about them as we talk about the type of inheritance of the
blood groups exactly.
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*Remember allelic exclusion> exclude one allele on the surface of a particular cell.




Chapter 7: The T- Cell Receptor
What about the T-cell?

The T-cell exactly follows the same format as those of immunoglobulins. So when
we talk about the T-cell receptor (TCR), how does it differ from that of
immunoglobulins and what are the similarities between TCRs and IGs?
Summary of Ch.6:
To make a specific anti body (AB with certain specificity) you
have to:
1. Build a light chain either:
- Kappa by select 1 V gene out of 35 & 1 J gene out of 5 plus
the constant
- Or lambda by 1 V gene out of 30 & 1 J gene out of 4 plus
the constant (of 4 types)
2. Build a heavy chain by select 1 V gene out of 50, 1 D gene
out of 25 & 1 J gene out of 6 plus the constant (of
different types; 5 isotypes; M,D,E,G,A)
Bearing in mind that changing the C without changing the V
will not contribute to the specificity only to the biological
function.
- The previous plus the somatic hypermutation and the
junctional diversity and the chance of which light chain
is going to bind to which heavy chain all contribute to
the Ig diversity & having about 10
11
different specificities
of Ig.

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**Differences between TCRs & Igs:
1) Immunoglobulins can bind to antigens freely and form immunocomplexes
while TCRs cannot do that. In TCRs the antigen has to be presented with a
class 1 or 2 MHC to the T cell receptor.
2) The TCR does not have what we call "somatic hypermutation" (a better fit
in the secondary immune response).
Also from Hayat archive
3) We have so many joining (J) genes that are associated with the TCR rather
than Igs. So, this will add up into the diversity, also you have Junctional
diversity.
4) The TCR is found in two polypeptide chains while in Igs we have four.
**Similarities between TCRs & Igs:
1) The outcome shape of both of them which produced by the
immunolglobulin super gene family is very similar.
2) The TCR goes almost through the same changes that occur with the Igs
diversity.



So
here we are talking about germline TCR genes which will form the two
polypeptide chains o and | for TCR 1 that form 95% of TCRs, and the two
polypeptide chains and o for TCR 2 which found in a little percentage. So we
have germline genes o and |, then gene rearrangement, the first one will act as a
light chain and the second one will act as a heavy chain. The second one; the |
chain will have the diversity (D) genes, both of them will have J genes, then we
have transcription and translation then we will have o and | polypeptide chains
then have a TCR assembly.
As a summary:
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1)You have o| , o then 2)you have the V(D)J, recombination, transcription,
translation and then this will code for the o| or o polypeptide chains and 3)then
the TCR assembly takes place.
In TCRs the number of genes that we are dealing with is much much more
compared to those of Igs. So we expect the diversity to be higher; around 10
^16

10
^18
different specificities of the TCR. So it makes sense that we need to have
more specificities of T cells because of the nature and function of these cells that
they are going to help T cells as well as B cells.
So if you look to the structure of TCR (below) you can see two polypeptide chains
o and |, while in immunoglobulin we have two heavy and two light chains (4
chains as total).
The specific part here -which we call it the variable region of the o and |- is the
upper part which is exactly like the variable region of the heavy and light chains of
Igs. Also we have to notice that the antigen that will fit to the specific region of
TCR must not be free; it has to be carried on MHC molecule.









So we have a dual type of specificity; the 1
st
one between antigen and MHC
molecule, and the 2
nd
one is the specificity between o and | chains. Then we have
the transmembrane region which is always part of the cytoplasmic membrane.



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Here we are talking about variable and constant regions the same as Igs. So what
is the difference here is that we have variable o and constant o for the o chain
also variable | and constant |. So the most specific part of this TCR is going to be
the area between the Vo and V| which is a relatively flat antigen recognition
region that interacts with peptide antigen and MHC antigen and this is the
difference between TCR and Ig.

This structure is for the extracellular portion of
TCR. So we have an extracellular portion and an
intracellular portion and here we are concerned
with the extracellular part. This is the most
variable region (the flat surface in the figure) that
we want the antigen and MHC antigen to fit into.
So the diversity is going to be the same; that it
has to be applied to the o chain that represents
the light chain and the | chain which represents
the heavy chain, and this is for TCR 1 while for TCR
2, represents the light chain and o the heavy
one. So the difference here is that the | chain is
going to have the D (diversity) genes and o chain
is going to have it as well.

So look here (at figure 7.3) these are the | and o chains which will form the TCR1.
The o will act as the light chain and it has around 80 different V genes and around
50 J genes, so you see how much diversity we will have while in Igs there are 5-6
genes (probably of J genes) as a maximum. While for | chain there are violet color D
genes, we have here two of them but they are variable in number; so they could
be more than that. Also there are around 50 V genes, 6 J genes and constant
regions.

Figure 7.3
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From Hayat archive
If you want to calculate the total probabilities of variations you require [V and J
from the o| and [V and J from the |] and you can multiply those plus the
Junctional diversity to see variation of the o| type and the same thing for the o
type.
For TCR 2 we have the (light chain) and o (heavy chain), o chain will have the D
genes while chain only has V genes, J genes and constant regions.

From Hayat archive
1) The o type is present mainly on epithelial surfaces, and can bind to the non-
protein. They dont require even antigen presentation for those. So:
2) The o type seems that they act as the first line of defense rather than the
o| type.







In this figure, look to the TCR and follow up the colors, you can see that the red
color represents the V genes, the yellow color represents the J genes and the
violet color represents the Diversity genes. You start from the germ line genes of
o and the germ line genes of | chain. You see in the o genes, there are around 80
V genes, many J genes in addition to the constant regions. While in the | genes
there are D genes. In the germ line genes of o chain, you can see that V5 & J2 are
selected, then they will be recombined to form the o chain and the same thing is
for The | chain in which here V3 and J1 are selected in addition to D genes and C
regions, then all of these genes will recombined to form the | chain.

Slide 22:
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I have just said that this is the TCR, and it does not accept the antigen which is in
yellow freely; the antigen has to be presented with class 2 MHC antigen on the
antigen presenting cell. If you remember when we talked about the antigen
representing cells, we mentioned the thymus dependent antigens, macrophages,
dendritic cells, inter vegetative cells as well as B cells.
And in all of these cells the antigen has to be taken in, processed and then mixed
with MHC antigen based on the specificity. As you see in the figure, this is the
specificity that should be applied with the antigen. Then TCR should bind both
MHC antigen (blue in color) and the antigen (yellow in color) which binds to it;
that the antigen when bind To MHC antigen they will form a new shape that has
to fit in TCR and we call this restriction which means the antigen has to be
presented with an MHC antigen to the TCR and TCR should fit both of these
antigens.
The complex of MHC antigen -which binds a peptide antigen- is the ligand for TCR.
And the top of the MHC molecule and the peptide form a relatively flat surface as
you can see in the figure. Also you can notice that only some residues of the
peptide antigen interact with the MHC molecule; so a part interacts with TCR as
specificity and part interacts with MHC molecule as a total. And I will keep
repeating all these information so frequently because we need it when we talk
about T cell activation and what really triggers the T cell to be activated.

Slide 23:


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Also here when we talk about specificity, this is the rule of the thumb* that we
always talk about in the thymus dependent antigens (adaptive immune response)
but sometimes some exceptions could take place: if you look to the combination
in the figure in the previous slide, you can see the flat surface of TCR binds to MHC
antigen and peptide antigen as I told you.
But sometimes some antigens we call them superantigens bind to the beta chain
without having anything to do with the specificity of TCR or MHC antigen, they
just bind to the beta chain in order to give a signal to the TCR then TCR is going to
be activated.
* So the rule of thumb is to have to be in combination with MHC antigens to induce
response, the exception is the superantigens.
We see that in some of the toxins produced by bacteria, such as Toxic Shock
Syndrome toxin that is produced by some strains of staphylococcus aureus
(group A beta hemolytic strep). The toxin is absorbed through the vagina in those
who use tampons (absorptive applications for the menstrual cycle), through that
it will be absorbed directly to the blood and it will interact with T helper cells in
this way (binding to the beta chain).
So as a result, T helper cells will be stimulated and start to produce cytokines(such
as TNF) immediately which lead to increase the temperature of the body and
cause tissue destruction. Also there is another example of these toxins which is
Erythrogenic Toxins; the cause of scarlet fever.
"Superantigen can bind directly to the beta chain of TCR without binding of MHC
antigen to the peptide antigen. So these antigens are not specific, which means
they do not have to fit into the specific part of the TCR, just when these
superantigens bind to the beta chain and between the MHC and the T cell is going
to be activated. Also you can notice from the figure that the MHC binding does
not involve the peptide groove".
Slide. 24:
18

Here you can see a presentation of class 1 and class 2 MHC. Class 1 MHC consists of
3 domains o 1, o 2 and o3. Another one which is for support called |2
microglobulin. Also you can see the specific part between o1 and o2 which has to
fit to the TCR.










Slide 25:








On the surface of the T cell is not just the TCR that is going to applied to the
antigen which presented with MHC but also on the surface of the T cell we have
other accessory molecules which play a major role in the activation of the T cell.
So these accessory molecules are essential for the function of T cells. For example
we have one of them here and is called CD3.

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We start describing the antigens which present on B cells or T cells by using the
CD system. CD we call it cluster of designation or cluster of differentiation
antigens and each antigen we give it a number (1,2,3,), then each one of those
CD1,CD2 ,CD3,. has a function and we have to know the function of each one as
time comes to describe that particular function.
CDs (CD1, CD2,) are detected by the Flow Cytometry and we call them
sometimes tumor markers if they are present in a mature cell where they should
not be present and vice versa; in an immature cell and so on. You will know more
about this when we will talk about tumor immunology.


Refer to the figure:
This is the TCR (o and | or and o), and this is an accessory molecule we call it CD3
which is a complex of 4 polypeptide chain -sorry we didn't get what the doctor said
about the structure of it but this is what is written in the book "it's a complex
comprising 4 different transmembrane protein chains , o, c, ,"- we have two zeta
and we call it zeta zeta*.
The function of the CD3 is to let the signal pass through. So when the antigen
binds to MHC then the CD3 lets the signal pass through, and as a result, there will
be activation of a biochemical pathway that will let some changes occur. So CD3
has a very important role in letting the signal pass through and if it is not found
nothing is going to happen.
*from the net; it seems that also there are 2 c chains.

We also have other types of accessory molecules which called CD4 and CD8. CD4
is present on the surface of T helper cell mainly but could be presented on some
macrophages and antigen presenting cells. And we will see how CD4 is also
important for the signal transduction and it recognizes class 1 MHC antigens.
The same thing applied for the CD8 which presents only on the surface of T
cytotoxic cells and it only recognizes class 2 MHC antigens in order to let the
signal pass through, and then activates the T cytotoxic cell.
We have other accessory molecules such as the CD11A and the Lymphocyte
Functional Antigens which play a major role as co-receptor molecules by
enhancing the signal passage and activation of the T cells.
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The End

Sub7an Allah, such a perfect organized system working on.. So don't worry; your
body is in safety! What's left is your soul, try to protect it
Done by: Sara Zuriqat & Mai Mazin