Today's lecturer is going to be on the generation
of antibody diversity.
As well as, there will be introduction to
immunoglobulin class switching.
If you remember from the last class, we have
already discussed how every different B cell,
which is naive has not encountered antigen,
has already on its cell surface specific receptor;
three-fourths of which is identical in all
the cells; only one-fourth is hyper variable;
and, it is this hyper variable region, which
is at the amino terminus, which is responsible
for specific interaction with antigenic determinants
on the different antigens.
We are capable of mounting an immune response,
which would make antibodies of at least 100
million difference specificities.
Now, this cartoon shows different B cells
and the recognition of a specific antigen
by clone number 4 would lead to activation
of this cell, proliferation in the presence
of into 4 and 5 secured by T cells.
And then, ultimately, differentiation to plasma
cells.
Just to recapitulate to your memory, the hyper
variable region responsible for antigen binding
on the heavy and light chains constitutes
any one of the several V gene segments, which
come in close proximity; in fact, joined with
any one of the J gene segments in the case
of the light chain gene.
And, in the case of the heavy chain gene,
it is V, D and J; addition of one more gene
segment in the heavy chain.
And, as I told you before, the heavy chain
gene product, which is the heavy chain of
immunoglobulin, gives much more regions for
binding to the antigen.
How it is that only one of the several J segments
present and one of the several V gene segments
present combined?
Now, this is because of the presence of special
recognition signal sequences, which I mean,
as I mentioned earlier, these are conserved
through evolution.
So, you have two sets of these recognition
signal sequences and it is a rule that the
combination or joining would take place only
between the two different RSS.
One, which is 1-turn RSS; this 1 turn corresponds
to 1 turn of DNA helix; or, 2-turn RSS.
Now, this is what would be required in case
of the light chain - lambda light chain assembly
as well as kappa light chain assembly.
Now, since there are three segments on the
heavy chain, the two RSS rules would need
to be modified.
And, this has been modified in the following
way.
The same RSS signal in this case, which has
been depicted, 2-turn RSS are present on 5
prime and 3 prime side of every D region segment.
And, the corresponding, that is, 1-turn RSS
are present on the 3 prime side of every V
gene segment and the 5 prime side of every
J gene segment.
Joining between D and J occurs first.
And, once this combination has taken place,
one of the several V gene segments are recruited.
Now, the question comes - how is this variability
of 100 different molecules to be synthesized;
100 different molecules, which has specificity
to recognize 100 million different molecules.
How are these generated?
All the information lies in the genome, but
T and B cells; both of them undergo recombination
with respect to their receptors.
And, this is how the generation of diversity
is brought about.
Now, there are 7 plus 1 different mechanisms
that go to providing this process for generating
tremendous diversity.
First, one is multiple germ line gene segments.
Second, combinatorial V-D-J joining in heavy
chain; and, V and J joining in light chain;
combinatorial association of recombined heavy
and light chain; junctional flexibility; P
region nucleotide addition; N region nucleotide
addition; somatic hyper-mutation.
And, last but not the least, which does not
contribute to the recombination with respect
to variability; yet, one can say, this also
provides diversity, because once the VDJ recombination
has taken place, then the recombined region
can combine with any one of the constant domain
region in the heavy chain increasing the diversity.
That means the same recombined V-D-J can associate
M and D simultaneously that happens on the
navies cell as well as any one of the other
constant domain gene.
Now, let us go to the first mechanism, that
is, multiple germ line gene segments.
I have on the slide, the estimated V gene
segments corresponding to the lambda light
chain in mouse, human, the kappa and the heavy
chain.
I would like to take the simplest one first,
lambda light chain.
You can see, in the mouse, there are only
two variable gene segments corresponding to
the lambda light chain in mouse, in human.
However, this has evolved to have 100 different
V gene segments.
Now, when I say different V gene segments,
please remember, we are referring to the sequence,
the nucleotide sequence, which is of course,
then corresponding to the amino acids.
Now, why in the mouse, there are only 2 V
gene segments?
This would mean that the light chain corresponding
to lambda is reconstructed.
It is important to remember here and I have
mentioned this earlier that 95 percent of
the immunoglobulins in case of mouse are associated
with kappa light chain and not lambda light
chain gene segment.
Now, let us come to the J gene.
In human, there are 6 J gene segments on the
lambda light chain, 4 in mouse.
And, now, this is something I would like you
to see that though there are 6 constant domain
genes corresponding to the lambda light chain
in human, and 4 constant domain genes in mouse.
All these are identical.
Now, kappa light chain because remember again,
the light chain would have only one constant
domain.
A kappa light chain - there are 300 different
variable gene segments in mouse; 100 in human;
5 J in both and 1 constant domain.
Heavy chain - interestingly, in the mouse,
there are 300 to 1000 variable gene segments;
that is really a very large number.
In case of human, it is 100 V gene segments.
But, the human heavy chain makes up by having
more diversity gene segments; there are 30
in all.
Mouse has 30, 6 J and 4 J in human and mouse
respectively, and of course, series of C,
that is, constant domain gene segments; each
one corresponding to the class of immunoglobulin.
So, if you have that many different V gene
segments, and I have told you, joining of
V-J or V-D-J in heavy chain is totally a random
process, and therefore, one can see that this
random joining is what gives rise to diversity.
And, if you do a mathematical calculation,
then if you look at just the mouse combinatorial
V-D-J joining, heavy chain and in the case
of light chain, V-J, then the possible number
of combination would happen in the heavy chain;
300 into 13 into 4, which would give to 1.6
into 10 to the power of 4.
Now, this along with the light chain, which
is 300 and 4, because of absence of D segment,
would give rise to 1.2 into 10 to the power
of 3 combination between these two combinatorial
associations.
Immediately then increases the permutation
to an extent, where you can have 1.9 into
10 to the power of 7; 1.9 times, I meant,
into 10 million of heavy and light chain.
So, let us go back and just think that we
have now very large number of V-D-J and combination
between any one.
Now, each B cell would have the capacity to
join any one.
So, randomly, you would have any one of J,
D and then V being recruited.
And then, again light chain, which is combined
separately; the heavy chain combined separately;
and, these two coming together after the translation
when folding takes place in the cell, combination
of this again would give for the diversity.
Therefore, we have covered now the first two;
that is, several of the gene segments, which
have the capacity to recombine at the DNA
level followed by combinatorial association
between the heavy and the light chain.
Let us come to the third, which is junctional
flexibility.
What is junctional flexibility?
Now, since we are talking about two segments,
which join together...
Now, if you remember, in the last class, the
mechanism of RAG 1 and 2 cutting the single
strand of DNA specifically at 
the RSS; the RSS, which are, if you remember,
hepatoma and the AT-rich nanoma, they come
very close in proximity, and this is done
by the RAG 1 and 2, which recognize these
signal sequences.
They bind and recognize and they bring them
together.
Now, cutting of the single strand DNA occurs
by RAG 1 and 2 precisely at the hepatoma.
Therefore, though you get signal joints, that
would mean, when the hepatoma, nanoma, the
RSS are deleted, if one sequences these deleted
products, one can see that the joining between
the hepatoma, nanoma, the signal sequences
are very precise; it is identical as seen
in pre-B cell lines, which are going on to
becoming B cells, where recombination is taking
place.
This is experimental evidence that shows that
signal joints are very precise.
But, when the coding joints join that it would
mean 1 D with 1 J or 1 V with 1 J or V with
D J. The coding joints always are imprecise.
And, we will come to how this happens in two
minutes.
You to see this, the coding joints, which
of four different cell lines, which are derived
from one pre-B cell line; this pre-B cell
line was now activated to start the recombination
process in to 7 at as in V where you have
the bone marrow providing into 7.
Now, the pre-B cells go on to recombining
the heavy and light chain.
And, when coding joints were sequenced, it
was seen, the several 1, 2, 3, 4 different
cell lines have different sequences.
This imprecise or difference in joining in
the coding joints can of course, be deleterious.
And, as seen in the last experiment,the coding
joints joining together can also change the
sequence, the reading frame in such a manner
that you may have the introduction of stop
codons.
And, this is what has happened in the 5s and
9s that were then looked at; other five, where
two of them, the recombination failed, because
of the change in the reading frame, stop codons
were introduced.
However, productive rearrangement happened
in 3.
Why it is important to know this is, because
remember, when we talked about the development
or the ontogeny of these cells, in the antigen-independent
phase, there are large number of B cells,
which undergo apoptosis, because of lack of
precise joining.
One 
of the reasons is introduction of stop codon
in the reading frame when coding joints were
joined together changing the reading frame.
Next, would be, that is, 4 and 5.
4 - the fourth mechanism in introducing diversity
would be addition of P-nucleotides; and, N-nucleotides
would be the fifth one.
Let us look at the addition of P-nucleotide.
I have a diagram in the next slide, but let
us first go to the theory part.
In this particular example, one of the D is
joining with one of the J and by the transesterification
reaction, RAG 1 and 2 have made a palindrome
sequence.
You have now in the coding sequence of the
D segment, TC; and, in the lacking strand,
AG.
Now, remember artemis, the endonuclease, which
is specific with respect to recognizing hairpin
structures.
Now, this protein, this enzyme binds to the
hairpin structure and can cleave anywhere,
because cleavage is not specific with respect
to the nucleotide sequence.
Now, let us see, in case artemis cleaves at
the hairpin junction between the D gene segment
and this hairpin structure.
Now, if the cutting is in the lagging strand,
closer to the lagging strand, then you would
get TC; look at the coding strand first; the
coding strand would have the TC, which was
originally there plus would get GA from the
lagging strand.
Similarly, if you look at J gene segment,
you have now cleavage occurring at the joint
after TA, which would mean now that the lagging
strand would get here; AT from the coding
strand.
GA has the different color.
GA and AT are not present in the sequence
of the coding or the lagging strand.
They are new nucleotides with respect to that
particular strand.
When joining takes place, you would have end
filling before the ligase activity.
And, you can see now, four nucleotides in
the coding strands, which are different from
those that were present earlier.
Therefore, now, these are unknown as P-nucleotide
addition.
This of course, as you can imagine, would
generate diversity.
But, this would also if the reading frame
now adds a stop codon midway, would also be
deleterious.
However, this is one of mechanisms of diversity.
Let us look at this unique system of addition
of N-nucleotides.
When the hairpin structure has already been
cut by the artemis as I already mentioned
and described in the P-nucleotide addition,
there is expression of a specific enzyme,
which again specific to lymphocytes.
Terminal deoxyribosyl transferase - you might
remember from my first slide in my first lecture
that TDT is the marker for B cells and T cells.
Terminal deoxyribosyl transferase - immediately,
you would realize that this enzyme has the
capacity to add new nucleotides.
So, at least with respect to P-nucleotides,
the information was available not necessarily
in that particular strand, but in the opposite
strand.
However, in the case of N-nucleotide addition,
in additions of nucleotides, which are the
catalyzed by the TDT and which are not present
at all, are there any preferences with respect
to the addition of any nucleotides?
It has been seen that there is no preferences
as such, but if you look at the number of
nucleotides, which are present in this particular
D-J joining, then, there is a preference for
G-nucleotides to be added.
So, now we have two more mechanisms: P-nucleotide
addition and N-nucleotide addition, which
would contribute to this diversity.
Now, all these are going to change the frame,
are going to change the amino acid sequence;
the information of which was a variable on
the coding strand earlier.
To look closely at the P-nucleotide addition
and to make it a little bit clearer, I have
a diagram here the coding strand and the lagging
strand, which is in dotted line.
Now, it is after all the coding strand that
matters.
And, let us look at the hairpin structure.
Now, there are two instances here.
The artemis is cutting the hairpin structure
at two different regions.
Now, in one, if the cleavage occurs closer
to the coding strand, then you would have
a much smaller nucleotide sequence from the
coding strand, which is present, and modern
half would go automatically to the lagging
strand, which I have not shown over here.
However, in case of two, if the cleavage occurs
on the hairpin closer to the lagging strand,
then you have a much larger fragment, which
would encompass quite a few nucleotides from
the lagging strand.
And, this would be new to the segment over
here, which could be let us say D. Cleavage
of the hairpin by artemis is not sequence-dependent.
Again, I would like to remind you, position
at which the hairpin is cut is variable leading
to variability in the sequence of the coding
joint.
Let us look at the Tdt gene expression.
When immunologists sequenced these V-D-J recombined
genes, they found that there were a few nucleotides
in the D-J as well as V-D; they could not
count for those on the lagging of the coding
strand.
And then, they realized that there was this
enzyme Tdt, which was adding new nucleotides.
So, after that, large number of sequences
were obtained and it was very clearly seen
that in fact, yes; there is nucleotide addition
in the heavy chain gene, N-nucleotide addition.
I would like to also tell you that N-nucleotide
addition happens mostly on the heavy chain,
because the expression of Tdt, terminal deoxyribosyl
transferase enzyme is decreased when recombination
of the light chain gene is happening.
There is a very nice experiment that was carried
out to show; I have told you in a few lectures
ago that people have been able to induce recombination
invent in fibroblast cells.
Recombination of the immunoglobulin gene or
rearrangement would not happen in any other
cell except T and B cell.
B cells in case of the immunoglobulin gene
and T cell with respect to the TCR, T cell
receptor; T cell receptor also has heavy and
light chain, etcetera.
The structure might be different, but in any
case, both of these undergo reorganization.
Now, one can induce reorganization of these
genes in fibroblasts by expressing a plasmate,
which codes for RAG 1 and 2.
So V-D-J recombination was induced in fibroblast
cells and only when these fibroblasts were
also transfected with the Tdt gene, N-nucleotide
addition was seen to take place.
So, definitely again, the relevance of Tdt,
the enzyme was very well demonstrated.
How many nucleotides can be added to the cut
ends?
And, as many as 15 nucleotides can be added
to 3 prime of both DH-JH as well as VH-DHJH
joints.
And, this constitutes CDR3.
This is very important to remember that the
CDR3 region is where N nucleotides are added.
So, highest diversity would be there.
N-region nucleotide addition - just like to
say a few more things with respect N; there
is no template specificity.
And, Tdt adds, like I said a little while
ago, dG residues preferentially.
Therefore, it has been also observed that
VD, DJ junctions are G-rich.
Again, like to mention that N-region nucleotides
are not added on the light chain, simply because
heavy chain assembly takes place first; Tdt
expression happens at that time and the level
of this enzyme starts going down when the
light chain assembly is taking place.
During the course of immune response generate
to an antigen, there is not only increase
in the titers of antibodies, but also the
affinity.
This was looked at a little bit closely.
How does this happen?
Now, remember this figure.
This is experiment that was conducted in a
rabbit injected with one particular antigen
on day 1.
Blood was collected from the rabbit every
other day or every third day, and then, the
titer of the antibodies generated in circulation
to the antigen was determined or quantitated;
so, the y-axis shows that; the x-axis shows
the days over which the blood was collected.
After the first response, when the titers
were seen to come down, the same antigen was
injected again, and also, at a third time.
Now, there has been slight change; this should
be here 60 and this 3 should be here.
That would show primary immune response, secondary
immune response and tertiary immune response.
This is what I told you already that you would
have a much smaller amplitude after the first,
which is absolutely expected and most of these
antibodies will be of IgM isotype.
You have then, memory cells, which will be
generated here.
And, when the animal sees the antigen again,
there is no lag phase.
Immediately, circulating antibodies start
appearing and the amplitude of the response
is higher.
Similarly, after the third injection of the
same antigen, again there is no lag phase
and you would see the amplitude, which is
much higher.
Now, when people started to look at the affinity
of the antibodies - this was only the respect
to the titer, it was interesting to observe
that the affinities of the antibodies increased;
and, this was every time.
In the third immune response, the affinity
would probably increase even a 100 fold or
a 1000 fold.
People started to look at the receptors on
memory cells versus those on the naive cells.
We know that the naive cell to a particular
antigen already has a predetermine sequence
of the hyper variable region.
The mutations were found in the memory cells
after the secondary and tertiary response.
So, there were quite a few experiments that
were carried out.
One of which I would like to mention over
here, because this gave a direct evidence
that though the variable region once formed,
that is, V-D-J, does not alter except for
a few mutation, which give the recombination
event a better advantage; how, let us see.
There were experiments that were carried out
where a hapten - why did these people take
hapten?
Hapten is a small molecule; it has an antigenic
determinant; it could be either a peptide
or it could also be a small molecule, such
as dinitrophenol.
Now, such a small molecule when needs to be
conjugated to a carrier protein for it to
become an immunogen; in this particular experiment,
scientists have taken phenyl oxyzolone, which
is a very good B cell epitope.
You get a good response to this very small
molecule, phenyl oxyzolone; molecular size
is very small.
It is conjugated and then to a carrier protein,
injected into mice.
And now, hybridoma was established from the
spleen of these mice on day 7, day 14, and
then, subsequently.
They got several cell lines, several hybridoma.
I have not introduced hybridoma, but I would
like to just mention, hybridoma are those
cell lines, which are generated by fusing
a B cell with a myeloma cell, so that the
B cell becomes immortal, the antibody producing
B cell.
Getting a cell line is always easy, because
several experiments can be done including
looking at the gene sequence.
Now, these hybridoma are monoclonal, and therefore,
all the cells that are generated in one particular
culture would be monoclonal.
So, every sequence would be identical in this
particular cell.
Now, there are large number of cell lines
that were sequenced with respect to the immunoglobin
heavy as well as the light chain.
And, as can be seen with the hybridoma that
were generated on day 7, neither the heavy
nor the light chain differ too much with respect
to the immunoglobulin sequence.
I would like to say that these small dots
and lines represent mutations in CDR 1, CDR
2 and CDR3.
I have not discussed these three regions,
but sufficer to say that both heavy and light
chains have three hyper variable regions,
even in the variable region.
And, as a name suggest hyper variable, you
have tremendous variability in these three
regions.
And, it is through these regions that antigen
binding takes place.
So, you can see almost no mutations seen in
the heavy and light chain in the first 7 days
after immunization.
Remember, now, the mice were immunized on
day 1; 7 days later, hybridoma were established.
Interestingly, when hybridoma were established
from spleens of mice, that immunized 14 days
prior, which would mean now that this would
be the secondary immune response.
You can see very clearly that the light chain
has accomplished or accumulated large number
of mutations.
Now, each one of these, for example, cell
line number 1 has two; the second cell line
has two; three mutations in 3, and so on.
This was quite also interesting.
Now, this could be only with respect to phenyl
oxyzole, but on day 14, it is a CDR 1 of light
chain seems to have accumulated.
In fact, if you look overall, in case of the
light chain, CDR 1 seems to have maximum number
of mutations.
Now, the mutations were really quite random
in case of the heavy chain in the CDR 1, 2
as well as 3 regions.
In the heavy chain, CDR 3 is coded for by
part of the day D segment and J. In case of
the light chain, CDR 3 is only J. You can
see the secondary immune response and you
can see the mutations have increased; you
can see by the number of dots.
You can see that the mutations are tremendous
when you come to the final tertiary.
Now, what is secondary and tertiary is day
7 and 14; the animal has been injected here.
Animal has been injected once more before
the secondary immune response and once more
after the tertiary.
You can see very clearly that these mutations
are accumulated in memory cells in response
to the antigen.
Now, look at the right side panel; I am sure
that is difficult to read, but I just I tell
you what does this show.
Now, the antibodies that were synthesized,
secreted by the hybridoma clones, but then,
assessed for their K d or the affinity to
bind the antigen.
And, what can we seen, let me read this out;
K d into 10 to the power of minus 7 moles.
So, this becomes 100 nano molar.
So, first the 
affinity of the antibodies is quite low, does
not change much from one cell line to another
in hybridoma that were established on day
7.
Immediately, when hybridoma were established
from mice on day 14, you can see immediately
an increase in the affinity 10 fold.
And this became, with this increased further
in the secondary as well as the tertiary,
by a 100 fold.
This was direct relevance.
And, this experiment has been reproduced several
times to show that during the generation of
an immune response, we have increase in the
affinity and this affinity can only happen
by receptor editing.
Considered by the sequence, if it remains
the same, there cannot be increase in the
affinity.
For the affinity to be increased there has
to be a sequence change.
And, the sequence change happens because of
the presence of an error prone polymerize.
And, this error prone polymerize gets activated
in memory cells.
Therefore, this will of course, then the receptor
editing as well as increase in affinity can
only happen in case of those B cells, which
are T cell dependent.
Remember, memory cell generation itself is
a T cell dependent phenomenon.
So, affinity maturation T cell dependence.
And then, may be if I have time today, I will
talk on isotype switching.
All these happen in T cell dependent B cells.
I would like to also tell you a relevant experiment
with respect to affinity of antibodies.
Several years ago, when people did not know
much with regard to these hemoglobin genes
and the affinity, it was believed that if
you injected milligram amounts of antibodies,
you would get a very robust immune response.
But, it is not.
In fact, I would also like to say, when one
is doing experiments with let us say, mice
and rabbits, rabbit would be let us say, even
1 kg and a mouse is a paltry 15 grams.
The amount of antigen that is injected does
not go by the body weight.
There is no pharmacological dose.
Immunological doses will still be in microgram
amount.
Typically, if you are injecting 20 micro grams
of immunogen in mice, in the rabbit, you would
inject not more than 10 fold, 200 micro grams;
but, not corresponding to the weight of the
animal.
There are two instances given over here; low
immunizing dose versus high immunizing dose
of the very antigen that I was talking about.
And, let us say, the animals are rabbits.
If the animal is injected with low immunizing
dose, what would happen?
Because the dose is very low, only high affinity
receptors of those B cells would recognize
the antigen.
Let say, this B cell has an affinity of binding
to the antigen to the order of 10 to the power
minus 11.
So, it becomes absolutely to fem to molar
range.
So, very low immunizing dose; let us say,
it would be something like 10 micrograms in
rabbit.
You would get an immune response generated,
but very few B cells would get activated in
response, because only the high affinity ones
would.
Only these would then get activated, proliferate,
differentiate and become plasma cells.
Maybe in certain instances, you may not detect
the presence of antibodies in circulation,
because the numbers may be very low.
At the low affinity receptor bearing B cell,
would not come into the picture at all, because
they would not mind.
So, there is no question of activation, proliferation
and differentiation.
Now, if a high immunizing dose is given, then
both B cells, those bearing high affinity
receptors as well as low affinity receptors,
would get activated differentiate, and you
would get a mixture of antibodies, which would
have both high affinity as well as low affinity.
So, effect of immunizing dose on affinity
maturation is important.
And, even if one starts with high dose of
antigen immunization one, subsequently, the
dosage can be decreased, so that one would
get mostly high affinity binding antibodies.
Now, let us go over this whole mechanism of
the establishment of diversity in the immunoglobulin
gene.
It is because multiple germ line gene segments;
combinatorial V-D-J joining; combinatorial
association of heavy and light chain; junctional
flexibility; addition of P region nucleotide;
addition of N region nucleotide; and, somatic
hypermutation.
Now, I would like to mention finally, that
we do have the V-D-J, which is already combined;
maybe it has undergone mutation, affinity
maturation.
This does combine with always the mu constant
domain, and simultaneously, with the delta
constant domain.
But, those B cells, which are T cell dependent,
can undergo class switching, and in them,
the recombined V-D-J can associate with also
any one of the other five classes of immunoglobulin:
the gamma, alpha, eta.
Since I have time, I will introduce to you
immunoglobulin class switching.
Class switching - I have not introduced again
already the five different classes of immunoglobulin,
but I would have mentioned when I was talking
about the genes that you have in the constant
domain; you have gene segments that code for
constant domain of IgM, that is, mu, delta
for D. Then, there are four gene segments
for the immunoglobulin gene: constant domain
G 3, constant domain G 1, constant domain
G 2 b and constant domain G 2 a.
Now, this is mouse, because in human, it is
the simpler; it is 1, 2, 3, 4.
So, human also have 4.
Then, follows; in the immunoglobulin gene
organization, you have constant domain eta,
that is, IgE, and then, constant domain alpha.
Immunoglobulin class switching is maximum
and very high during the naive cell generation.
Remember, in the ontogeny of B cell, IgM is
the first receptor that gets suffice expressed.
Now, by alternate splicing, IgD is also simultaneously
expressed.
So, during this, though this is not class
switching totally from one to the other, there
is alternate switching.
And, this is, like I said again, by alternate
mRNA splicing, that you have information of
IgM and IgD of naive cells.
Now, class switching is remarkably high in
the first instance and remarkably high during
the secondary immune response when IgM switches
to any one of the classes immunoglobulin.
Classes again would only be referring to the
constant domain genes.
Isotype switching occurs in germinal center,
1 week after immunization.
You have already been familiarized with the
secondary lymphoid organs and you already
know what germinal centers are.
And, this is where you have memory cells;
you have T, B and macrophages cells.
And importantly, memory cells of B cells,
which 1 week after the primary immunization,
if the antigens still persist, then there
is activation of memory cells and there is
isotype switching.
So, the memory cells, which would be IgM...
One thing, which I missed telling you all
earlier that though we have IgM and IgD, both
the immunoglobulins on the naive cell, IgD
is almost never expressed; and, the only time
once this IgD is in myeloma cell.
So, there is no IgD in circulation of very
low amounts.
It is always IgM, IgG, IgE, IgA; mostly, IgM
and IgG and IgA.
Now, 1 week after the primary immunization,
isotype switching takes place and this isotype
switching is absolutely requires CD40 receptor,
CD40 ligand interaction.
Again, I would like you to remember that CD40
receptor is present on B cells constitutively,
but require the presence of CD40 ligand, which
is made by the T cell for certain processes
of activation.
It is this interaction which now tells the
cells to produce the cytokine receptors and
also gives the progression signal to the B
cells for proliferation.
If there is no CD40, CD40 ligand interaction,
no class switching takes place; also, no memory
cell generation.
This has been shown by conditions in human,
who lacks CD40 ligand; there T cells cannot
make CD40 ligand, and therefore, there is
no CD40 receptor ligand interaction.
Therefore, no memory cell generation, no class
switching and no affinity maturation, because
this process is totally T cell dependent.
Isotype - the same variable, again and again
I would like to mention this that it is already
combined V-D-J, which now can associate or
disassociate from the IgM and associate with
any one of the isotypes.
Forget the IgD, because in fact, there is
no switch region between these two.
Isotype pattern varies according to antigens.
Now, I have another slide, which goes a little
bit more detail for, but suffice here to say
that, for example, why do I say isotype patterns
vary according to antigen?
Antigens such as viruses usually induce IgG2a
type of immune response; why, because of the
fact that IgG2a can bind to a specific receptor
on the T cells, which allow now antibody dependent
cellular cytotoxicity.
Helminth antigens - they induce IgE type of
an immune response or IgG2b.
I will be dealing with this a little later.
Carbohydrates on the other hand or cell wall
antigens typically induce IgG3 type of an
immune response, because IgG3 10 crosses placenta;
IgG3 can fix complement better, and the other
isotypes.
Now, of course, one can look at it from the
point of view that the element response to
bacteria can be conferred to the fetus.
And also, because IgG3 can fix complement,
these bacteria or pathogens can very well
eradicated even
Just introduced to you this recombination
event that takes place during class switching;
this also of course, again happens at the
level of DNA.
So, let us look at recombination.
Let us first look at the mechanism, how does
this happen.
Now, very clearly you can see, VDJ has already
recombined and fine waiting over here.
You have now, on the 5 prime side of the constant
domain gene segments, which are in different
colors switch region...
There is no switch region, please note here,
between the constant domains of mu and delta.
Therefore, you can have RNA splicing and alternate
expression on the naive cell.
Now, switch region, mu, needs to combine with
any one of the switch regions for the isotype
switching.
How does this happen?
Let us say, in this particular case, now you
remember, the organization of the constant
domain is exactly the way I have mentioned
earlier.
And, this does not change.
So, you have now this cell that is going to
undergo class switching once to switch from
mu, that is, IgM to IgG2b.
Now, this happens by the joining coming together
of the switch region genes come in cross proximity,
and then, you have the deletion of all the
sequences, which were in between the m and
the IgG2b.
This recombination between the switch region
S mu, that is, for the mu or the IgM and gamma2b.
Now, during transcription, again you will
have RNA and then you have the matured transcript.
Along with this, you will of course, have
this interesting structure.
This is the path that has been thrown out
of the DNA sequence.
You have, just like in the other strand that
is retained, you have a part of the switch
mu and a part of this switch gamma 2b, which
have joint, and this is part, which has been
deleted.
And, you can see the constant domain mu, because
now that cell can no more make IgM; neither
IgD, IgG3 nor IgG1, because all these are
in between the switch regions.
All these are between the switch regions mu
as well as switch region gamma 2b.
So, you can just visualize very well that
you have the switch regions that come together;
the recognized come together.
Then, you have cleavage.
So, you have a part of the switch region now,
which is still in this sequence and a part
which is here . Now, the same thing would
happen if the switching has to take place
between mu to let us say, IgE or mu to IgA.
Now, also look at something interesting that
if the cell has already undergone switching
from mu to IgG1, then this memory cell can
further switch from IgG1 to let us say, immunoglobulin
IgG2a; or, what is not shown here is immunoglobulin
alpha.
But, of course, since this part would be deleted,
the cell can no longer go back to making these
isotypes.
So, once again, there is cleavage and throwing
off or deleting an entire sequence.
In this case, it would be the constant domain
genes.
Now, I would like to stop here.
And, in my next class, I would like to talk
about what brings about this recombination,
is it known?
What are the different proteins, which bind
to this?
What induces?
How much is known with respect to class switching?
What can be these factors that govern?
Obviously, one can imagine and these factors
would be somehow associated with the kind
of the antigens that go in, because it is
antigens finally, which determine which kind
of immune response with respect to the isotype.
I already given example of viruses, which
induce IgG2a type in case of mice and IgG2
in case of human; whereas, helminthes, what
switch IgM to immunoglobulin E. Obviously,
then, the immune system has been involved
in such a manner that the immune response
should help to eradicate that particular foreign
object in the body.
Therefore, there should be generation of some
transcription factors; there should be generation
of interns, some proteins by the antigens
themselves; the immune response that is seen
in these antigens, which would culminate in
the production of those kind of immunoglobulins,
which would be highly effective.
So, next class, we will be looking at the
mechanism of this class switching further,
and also, looking at the immunoglobulin gene
regulation; what actually regulates all these
processes; are there any genes, which are
above RAG 1 and 2 and what these are.
Thank you.
