So, today we will be discussing some remaining aspects about MHC class 1 assembly and then
go on to discuss MHC class 2.
So, this is just to remind us, that you have T cells and they are activated by MHC peptide,
which is present on antigen presenting cells. We did discuss this aspect about MHC restriction.
What is, MHC restriction, the T cell receptor recognizes MHC, a particular MHC and the peptide
complex. So, the MHC is usually, that of the self and so they are restricted by self-MHC,
which means, the peptide binds to self-MHC and this is the altered self one that is recognized
by the T cell receptor. Now, this aspect is important in T cell activation,
which occurs peripherally, but it is more or it is also important during thymus selection.
So, for example, where do T cells learn to recognize the self-MHC? This learning process
is done in the thymus and so therefore, if you do not have MHC, you do not have T cells
because the learning itself is not done; so that is a very important aspect, that student
should be aware of. This TCR for T cell selection in the thymus, you need MHC. If there is no
MHC, you do not have T cell. So, for example, if there is some problem
in, let us say, MHC class 1 expression, you will not get CDAs or for that matter, if there
is some problem in MHC class 2 expression, you will not get CD4s. So, it is a very important
aspect, that you should be a sort of of course, in the periphery once the T cells get activated
by APCs, then it will result in IL2 and cytokine production, T cell proliferations, primarily
by, by CD4 positive cells and this interaction also results in the differentiation of CDA
positives into CTLS and then, ultimately killing of the target cells or the, or the ones representing
the MHC by the cytotoxic T lymphocytes. So, this is a, this is a broad over view of
what we had discussed in the past few lectures and if you, just to remind you about the structure
of the MHC molecules. Slide correction done here.
Now, MHC class 1, you have heavy chain and it consists of 3 domains: alpha 1, alpha 2,
alpha 3. You can see this, the alpha 3 domain is interacting with beta 2 macroglobulin,
which is very important for the assembly of MHC class 1 molecules. So, MHC class 1 molecules
needs beta 2 macroglobulin and the peptide is localized, the peptide binding groove is
localized in the alpha 1 and alpha 2 domains, which are present over here and we had talked
about some key residues, that are important in binding in peptides. These are known as
anchor residues and this is typical of, of MHC class 1 molecule and these anchor residues
are located in this groove. Now, MHC class 1 molecules, you are, consist
of 2 MHC class 2 molecules, sorry, consist of 2 chains, the alpha and the beta, and you
can see in the MHC class 2, the peptide binding groove consist, is made up of the alpha 1
and the beta 1 domains. So, the, the residues, there are, importance for binding are present
in the beta 1 and in the alpha 1 domain of the MHC class 2 molecule. Just to remind you,
that the MHC class 1 recognizes smaller peptides, about 8 to 10 amino acid peptides in MHC class
2. There are much longer, 18 to 20, you know, and sometimes even larger, so and as a result
of which you can see the MHC class 1. What is shown over here is that, the ends
are closed, that the peptide is closed, that is why, you cannot have longer peptides, whereas
the crystal structure of MHC class 2, as shown, that the ends are open. So, that is why you
have the peptide that can, actually you, knowing which, which, so, since the ends are open,
the peptide can sort of be a, be a lot, lot longer.
I am just to go over the basic model of MHC class 1. You have the intracellular antigens
or proteins, that are, are generated within cells or in case of one infection, you have
antigens, viral proteins, for example. So, these are degraded by the proteosome and the
peptides that are generated in the cytosol need to be translocated into the ER. Why ER,
because ER is the place where the MHC class 1 molecules assemble. So, this, this translocation
from cytosol to the ER is done by transporter associated with antigen presentation and processing,
we know as TAP. So, the TAPs are localized in the ER and these
are ATP dependent and so, the peptide ends up over here, and meanwhile, MHC class 1 molecule
are synthesized over here. MHC class 1 plus beta to micro ohm, they are synthesized and
the 4 over here, what is known as the peptide loading complex, the PLC, and the peptides
get loaded on, and then, then the MHC molecules there, there, there is, there is a structural
conformation changes in the MHC class 1 molecules. They are more stable and then they traffic
on to the cell surface by vesicular trafficking. So, the pathway is quite clear. So, you have
endogenous proteins, that are degraded by proteosomes and then you have peptides, and
then you have TAPs and then over here, these are peptides bind to MHC class 1 beta 2 macroglobulin
and then traffic on to the cell surface.
Now, I think, what we should do as an exercise is to try and understand, what would happen
if you do not have beta 2 macroglobulin or you do not have TAP? You see, now just to
remind you, that the MHC molecules are polygenic; both MHC class 1 and class 2 are polygenic.
So, you have different forms. So, even if you lack one, may be, there is, the other
one can be expressed. So, it is difficult to get a complete loss in MHC class 1 genes
because there are, there are so many and it is polygenic, and you are inheriting some
from the father, some from the mother, but what would happen if you do not have assembly
factor; I think, that is an important point. So, this is what was a, what is study. So,
beta 2 macroglobulin and as was shown previously, beta 2 macroglobulin binds to the heavy chain
and its importance gives its, gives its, gives it a stability. So, in fact, the trimer, which
the heavy chain, the beta 2 macroglobulin and the peptides are, required for proper
structural integrity of the MHC class 1 molecule. So, in the beta 2 macroglobulin knock outs,
so if you do not have beta 2 macroglobulin, you know, humans that do not express beta
2 macroglobulins or mice, that do not, are genetically engineers, such that they do not
express they are beta 2 macroglobulin, what happens is you generate low, unstable MHC
class 1 molecules. Also, because you have such a drastic reduction in MHC class 1 molecules,
you have very reduced CD8 positive T cells because the, the, the, the CD8 positive cells
are not being selected in the thymus and so, that is a very important, important aspect.
So, similarly, in TAP 2, what would happen is if you do not have the TAP knockouts, you
would generate, again, low MHC or unstable MHC, primarily because the trimer is not properly
formed. And again, you would have reduced CD8 positive T cells, again for the simple
reason, that, that again, selection in the thymus is not occurring because of problem
with the MHC peptide complex or, or lack of expression of the surface MHC peptide complexes.
Now, in the previous lecture, we had mentioned about a cell line known as a RMAS and RMAS
was critical in, in, in our understanding of the requirements for the surface MHC class
1 expression, but at that time problem in RMAS was discussed and ultimately, what, what,
what was found out is, in RMAS there is a problem in TAP 2. So, for some, for, you know,
the mutation results in TAP 2 not being expressed, as a result of which you have unstable MHC
class 1 molecules; low and unstable MHC class 1 molecules.
We had also discussed another protein, known as tapassin or this is a protein, that is
associated with TAP or TAP associated protein. And now, in, in this case, again you have
low MHC class 1. Now, tapassin apparently has a sort of editor function. So, it prefers
loading of slow off rate peptides, that means, the slow off rate means, that is, it is allowing
for high infinity peptides to be loaded on; so, it has an editor like function. Similarly,
in MHC class 2 molecules, we will see a role for DM and that is something we can discuss.
So, this is, this is a, so tapassin has an editor like function; it, it, it prefers loading
of slow off rate peptide. So, the slow off rate would mean that these peptides have,
have, so they come up slowly from slowly off, from the MHC class 1 molecule. That means,
they bind fairly strongly and so the structural integrity of these peptide MHC complexes are,
are much stronger or, or better. So, this was shown actually in, in mice, that lack
tapassin. So, we will start again and we will start
off with tapassin. Now, now, I had mentioned about tapassin and tapassin is a TAP associated
glycoprotein and, and what, what it does is, it, it helps, it increases the half-life of,
of TAP s and it helps better loading of MHC class 1 molecules with peptides. It turns
out it has an editor function, which means, it prefers loading of slow off rate peptides.
So, what this means is, that you have peptides that come off slowly from MHC class 1 molecule.
So, in the presence of tapassin, what tapassin does is, it helps MHC class 1 molecules have
a, and the peptides have a greater structural integrity. And in fact, this editor function
the tapassin has is, sort of, similar to an editor function in MHC class 2 pathway, and
that is primarily seen with the DM molecule. So, clearly, you know, it is an, it is an
important aspect. Now, we had also talked about the proteosome
subunits Lmp2, Lmp10 and in these single knock outs of Lmp2 or Lmp10 result in less CD8 positive
T cell numbers, but MHC class 1 expression is not greatly affected. You just have fewer,
some fewer, less number of CD8 positive cells in the Lmp7 knock out, though you have, the
MHC class 1 expression is low, but it is not as severely affected as in the TAP 2 knock
outs or in the, in the beta 2 macroglobulin knock outs. So, you have different consequences
of this. It is important for students to think in terms
of genes and what would happen if there are mutations in these genes and what would be
the phenotypes or what would be the function? So, actual function gets shown by these methods.
So, it is something, that student should be, should be very well versed with and aware
about.
Now, apart from the major molecules, that we discussed, beta 2 macroglobulin, TAP, tapassin,
there are other molecules, that play an important role and that is primarily because the assembly
of MHC class 1 molecules occurs in the ER and in the ER, there are other machinery,
that are also involved in this. So, some of these otherwise are, are 2 proteins, calnexin
and calreticulin. Now, calnexin and calreticulin are certainly not primarily associated with
the MHC class 1 pathway, but they play a role in, in protein maturation in the ER and as
the consequence of that, they are, they are also involved in MHC class 1 assemble pathway.
Calnexin is an ER membrane protein and it was one of the 1st chaperone shown to, bind
to, to a free heavy chain. In fact, it is, as, as MHC class 1 is synthesized, free, free
MHC class 1 heavy chain binds to a calnexin. However, in cells, that lack calnexin, there
is no defect in MHC class 1 assembly. Now, even though calnexin binds to it, it has no
obvious functional role. Now, calreticulin is similar to calnexin except
that it is luminary present, it does not membrane bound and in the ER, what was, what was shown
is calreticulin, you know, lacking cells, there is some defects in MHC class 1 assembly.
Of course, those defects are not as much as shown in the, in the, primarily one that we
discussed, which is beta 2 macroglobulin, TAP and so on. What was shown actually, with
MHC class 1 molecule are not loaded with optimal peptides, that means, the peptide binding
on it is not optimal. What actually happens is calreticulin is a part of peptide loading
complex and so if calreticulin is not there, perhaps the peptide loading is not that efficient
and which is why it results in this phenotype. What is important for students to understand
from a cell biology perspective is that both calnexin and calreticulin are important protein
folding in ER as they bind to mono-glucosylated N-glycans.
So, you have to think, you have to remember, that MHC class 1 molecules, these are glycoproteins
and as they are being folded, if the, if in some cases the folding is not proper, you,
you have a glucose being put on, on top of the terminal mannose residues and these are,
these are recognized and , that the folding is not proper and both calnexin and calreticulin
are part of molecules, that bind to these miss folded proteins and helps them fold properly,
and that is why, I have said, that you should be, sort of, aware of, of these things. What,
what happens is while you are studying immunology, you cannot study immunology in isolation.
It is part of, you know, cell biology, physiology, all these are, are integrated in, in, in this
molecular biology or it is integrated and you need to understand it and that is why,
I have put this, this part in about calnexin, calreticulin and the fact, that they bind
to mono-glucosylated N-glycans.
See, some other molecules are also important, for example, the ER 57, which is a thiol-oxiodoreductase
and this belongs to the protein disulphide isomerase family. What it helps, what it does
is, it, it helps in the proper disulphide bond rearrangements in nascent glycoproteins
as and that are recruited by calnexin or calreticulin. So, it is part of the better folding, ER folding
apparatus in the, the protein folding apparatus in the ER, that, that you can, you can appreciate.
Now, another aspect that I think we should discuss is the role of TAP polymorphisms.
Now, I had previously, when we were discussing MHC, I had said, that MHC class 1 molecules
are there, are there, are different aspects. What MHC class 1 molecule 1 is the localization,
they are H2 or H1 allocated, they are interferon-gamma inducible or cytokine induced because then,
you have an amplification of the immune response. The other characteristic is they are polygenic,
several genes and polymorphic, your several genes, that are encoding and within the genes,
there are variance, within, within these genes in different people. So, it results in polymorphisms
and these polymorphisms are useful because it helps them bind to different types of peptides.
Now, what is the role of TAP polymorphisms? Now, what was, what was, what is known is
that the mouse and human MHCs are highly polymorphic, but human and mouse TAPs are not, not very
polymorphic. There are some polymorphisms, but nothing at the level of, which the MHC
molecules are polymorphic. Now, the Rat MHC or the RT1 is the Rat MHC class 1, is not
polymorphic, but the Rat TAP 2 is polymorphic and here, here is a very interesting observation.
So, RT1A, which is this particular haplotype, the Rat MHC class 1, now this together with
Rat1 TAP2A results in efficient assembly of RT1. That means, you get properly loaded and
you have good surface expression of RT1. Now, if RT1A is associated or as you put it in
cells, that contain Rat TAP2B, which is a different form of, of TAP, TAP2, it results
in inefficient assembly of RT1. Now, why do you think this would be? Can you just think
a little bit about it? Now, it is possible, that, that this particular
Rat TAP2 translocates peptides, that are not optimal for binding RT1A. So, you have peptides,
that are, that are coming in to the ER, but those peptides are some optimal for binding
RT1A and whereas, the peptides, that are translocated by or the preference shown by, by Rat TAP2A
is the type of peptides, is more, are more the types, more of the types, that RT1A prefers.
So, you can see, as a result of which, so what happens here, in this case RT1A sets
in the ER because is not getting properly, proper, is not getting right type of peptides.
In the absence of the right type of the peptides, the folding is not proper and since the folding
is not proper, the, the, those molecules will not aggress to the cell surface. So, I hope,
this aspect is understood and as I said, it, it, it, you know, you understand the cell
biology of MHC much better if you understand the cell biology of protein trafficking, vesicular
trafficking, so on.
Now, the other aspect that I thought I should cover is, is that MHC, MHC, TAP proteins and
viral products. Now, as, as I mentioned previously, viruses are present in cells and they synthesize
their polypeptides, you know, in the cytosol, so that they can make more of their numbers.
Now, the, the immune system is such that you have mechanism by which these proteins, that
are generated in the cytosol are, are broken down in the peptides and these peptides are
presented on MHC class 1 molecule and they are expressed on cell surface. This is a way
for T cells to, sort of, peruse and get a feel for what type of, what is happening inside
cells. Now, and you can see, that one of the ways
by which and since the CD8 response is, is, is dominant, especially for virally infected
cells, viruses that synthesize proteins or have recruited proteins, that will inhibit
MHC, may be advantageous for them. It is a double edged zone because on one hand, it
might save them from CD8, but is MHC class 1 expression is lower, it make them sensitive
to incase cells. But perhaps, there is some sort of a balance in that and from our point
of view we need to understand what are some of the factors, that, that play an important
role. And over here, what was found is that there are several viruses, which encode proteins,
which inhibit the MHC class 1 assembly pathway. And as I said, now, over here, we need to
understand if you, if you want to block MHC class 1 surface expression, it is better to
target some of the assembly factors because then that will affect a larger number of MHC
molecules rather than targeting the heavy chain by themselves alone. So, it is much
smarter to, to target the assembly factors. So, again, that is something, that you should
try and understand. So, I will give you some examples. So, ICP47
is a, is actually encoded by the herpes simplex virus and it binds to TAP on the cytosolic
side and inhibits peptide binding. On the other hand, US6 is a product of the human
cytomegaloviruses and it binds TAP and inhibits TAP activity by preventing TAP hydrolysis,
without affecting peptide binding. You can see in these 2 molecules, that these are 2
proteins encoded by 2 distinct viruses, they do the same job, which is inhibit TAP, but
the way they do it is different. ICP47, it inhibits peptide binding whereas over here,
it inhibits ATP hydrolysis without affecting peptide binding.
So, you should see, different viruses have evolved in different ways and by which they
are trying to outsmart the immune system or at least MHC class 1 pathway by adopting various
strategies. So, I think, it is an interesting aspect of, for, for students to think about
and so the, you have some more examples. MK3 proteins of mouse herpes virus binds to TAP
and it, sort of, targets for a, for a degradation. The E1619 protein of adenovirus E3 disturbs
interaction of MHC class 1 with the peptide loading complex, the PLC, as I said and impairs
peptide loading. So, you have different mechanisms, where,
which these virally encoded proteins, they affect MHC class 1 peptide interactions and
loading, and as the result of which, the surface expression of MHC class 1 will be affected.
Now, now, one of the other differences between MHC class 1 and this is actually an important
difference between MHC class 1 and MHC class 2, is that the MHC class 1 is present on almost
all or most, most cells express MHC class 1. However, MHC class 2 expression is restricted,
which is present on APCs, on antigen presenting cells and, and these are, you know, B cells
macrophages, dendritic cells and thymic epithelial cells, so on, and these are, and this, this,
this bunch of cells is called the professional antigen presenting cells and interferon-gamma
increases the expression of MHC class 2 in, in antigen in APCs and also in non APC's sites
of infection.
Now, we can discuss a little bit about these antigen presenting cells, primarily because
of, because of MHC class 2, which is, shows a restricted cell type expression. So, macrophages
for example, they have different type, different organs, so they go by different names. And
this is the name given to ones, in the liver are known as Kuffer cells, these are peritoneal
PECs, peritoneal exoratecell, which are present in the peritoneal cavity, you have alveolar
macrophagocytes, alveolar macrophages are present in the lung and in just large particles
because you know, we take in so much dust and so on. And they produce cytokines, they
help in lymphocyte activation and they express costimulatory ligands, and these are aspects
that we will be discussing in the T cell activation part. The other types of antigen presenting
cells are, are, are B cells. Now, B cells are particular in the sense because they can
endocytose the antigen with membrane immunoglobulin, that is present and that is what make them
distinct from macrophages, in, in that sense, the microphages are going to be nonspecific.
So, they can use FcR or C3R or the complementary receptor or mannose phosphate receptor that
is going to be nonspecific; antigen specific endocytosis is done by B cell. So, in, in,
in, in this respect, the B cells are somewhat distinct from the other type of APCs and so
the, the, so they can process this antigen as an endocytose, and they can activate T
cells and they are, and T cells on the other hand, they activate B cells. And so, they
express these molecules and you have good interaction between T and B cells, which leads
to a good adoptive immune response. Remember, the 2 major players in the adoptive immune
response are B cells and T cells.
Perhaps, the most physiological antigen presenting cells are your or dendritic cells and these
are present in the skin. In the skin they are known as Langerhans cells and basically
the, the, once they get the antigen, they enter into the lymph nodes and, and they initiate
the T cell activation pathway.
Now, how does one study MHC biogenesis? One critical technique, that has been used in
this process is something known as Pulse-Chase experiments. I hope you are somewhat familiar
with it. So, what do you mean by pulse; what do you mean by chase? So, what happens in
the pulse part is that you pulse with a radioactive tracer and in for the type of experiments,
that we discuss of, mainly cell biology experiments, you use a certified methionine or cysteine
because these amino acids would get incorporated in most proteins, and at least sulphur containing
proteins and then, so, so that would be the pulse. So, what happens, that you would label
them for a short pulse, so it is always done in a short this thing, 5 to 30 minute, you
wash off the excess label, the label that is not incorporated. So, incubate the label
with the cells and so this gets taken up and it is incorporated by, by proteins that are
newly synthesized. So, newly synthesized proteins will incorporate
this labeled amino acid in it and then, you remove, wash of the excess label. So, now,
these are your cells, that are pulsed. What you need to do is chase and you chase with
a cold or excess methionine cysteine. So, that is no fresh incorporation of the radioactive
label into your proteins and, and then for, and this can be done at, for different period
of times. So, you chase for different periods of time and then you solubilize the cells
with the detergent and immunoprecipitate specific proteins. So, that is basically the theory
of pulse and chase and this is a very powerful technique.
What we will do is we can discuss some, some experiments with you, I think it is very important
because by discussing experiments, that is how you are going to be learning, you know,
a little bit more about these issues. So, pulse-chase is used to study biogenesis
of molecules, precursor-product relationship. So, for example, immature MHC class 1 molecules
are found in the ER/cis-golgi and you can distinguish immature class 1 from the mature
class 1 by the sensitivity to Endo H, which is a, which is an enzyme. So, the Endo H ones
are, Endo H MHC molecules are, are immature, whereas once they mature, once they have the
Salic acids, all sort of put on, on the, on the, on the MHC class 1 molecules, then they
become Endo H resistant. So, as a result of which, you can distinguish them immature and
the matured one; so, something very important. So, I think, you can think of experiments,
that, that are around this particular thought process. In fact, I mean, these are experiments
that were actually done to show the, the, the pathway is involved in MHC class 1 and
MHC class 2. Now, so, 2nd example, that I would, that I
would come up is, you know, as I said, once if you have that binding to MHC class 1 molecules,
then they become more stable and they can be detected with specific antibodies. So,
you know, you need to think of or design in experiments, how you would test the effect
of, let us say, produce of, specific inhibitor on MHC class 1 formation. Now, what are, what
are produced in specific inhibitor? So, this inhibits proteosomes. Now, remember, proteosomes
are the ones, that, that generate the peptides in the cytosol. So, if you inhibit, this peptides
would not be generated, peptides are not generated, then MHC class 1 molecule, there are no peptides
basically. So, it will be difficult for MHC, for class
1 molecules, to regress to the cell surface or an in, even if they expressed, they would
be unstable. So, they would be quickly internalized. So, that is some aspect that you can think
about, but you know, pulse-chase experiments are the type of experiments, that people have
done to address these issues.
Now, we will, we will start off with MHC class 2 right now. Now, MHC class 2 molecules, once
they bind to peptide, you know, they are extremely stable. So, how would you demonstrate this
finding? Again, we will, we can use the pulse-chase experiments for this, for this, for this question,
to address this question. So, let us say, you have labeled methionine
cysteine cell extract, you can, immunoprecipitate MHC class 2 molecules. Now, what you can do
is, after you have immunoprecipitated, so basically you have labeled type of extract,
you will have beads; that beads are important because you are, sort of, a matrix and to
these beads antibodies are attached, which will recognize the MHC class 2. So, the MHC
anti, so these antibodies will recognize MHC class 2 molecules. Remember, once you are
labeling your protein, all protein that are being synthesized will be labeled.
So, from this pool of labeled proteins, you need to separate the protein that you are
interested in. So, the reagent, that is used, is a particular reagent antibody. So, you
can use, you know, specific MHC class 2 antibodies to remove MHC class 2 from the spool of label
protein. So, that is, that is what is done. So, once you, you have it, now there are 2
ways by which you can treat, one is you do not heat the sample before loading on the
gel. So, and you just, you put it up on a, on a SDSP page and, and sort of, run it. And
the other is you heat the samples before you load.
Now, when you heat the sample, what will happen is, remember MHC class 2 molecules consist
of alpha and beta chains. They will fall apart and the peptide will be gone because you will
not be able to see the peptide in the gel. You will see, you will see the MHC class 2
and MHC class 2-alpha and class 2-beta. Now, if you do not heat, then the MHC class 2 molecules
will remain together along with the peptide and so they will run as a complex of MHC class
2-alpha, beta and they are held together. They will migrate with the higher mobility,
then the ones where you have heated. So, it is an important experiment to show,
that, that the MHC class 1 molecules, the MHC class 2 molecules are, are, will be held
together in the absence of heating. And now, if you, if you do these sort of experiments
from cells in which there is a mutation, that FXMHC class 2, what, and if the FXMHC 2 molecules
are unstable, then what will happen or they are bound to peptide set, are not very stable,
what will happen is, even in the absence of heating, the MHC class 1, class 2-alpha and
beta peptides will fall apart. And in fact, these are the sorts of experiments that were
done to show, that you know, there are some molecules that are important for MHC class
2 stability. And in fact, if you can translate this cell with some of these assembly factors,
then you find, that yes, you know, you have regain back the ability of MHC class 2 molecules
to sort of being associated with peptides and migrate as a complex; very important experiments
and that is why, I initiated this part of talk with the pulse-chase experiments.
So, we are, we will discuss MHC class 2 assembly. Now, as MHC class 2 is synthesized, the alpha
and beta chains, they come together, they bind and they associate with a particular
protein molecule, known as invariant chains. Now, why is it called invariant? You see,
because for the people in the MHC field, they are so used to polymorphism and polygenic
proteins, that MHC class 2 alpha and beta are obviously, polymorphic, polygenic and
they associate it with an invariant protein molecules, and then hence, they were named
invariant. The other distinguishing aspect about the invariant shape is that it is not
encoded in the MHC; it is encoded in a non-MHC location. So, but anyways, once it is synthesized,
it comes together, what this invariant chain does is that it forms a trimer of trimers.
So, basically it is a nonomer and you will have 3 MHC class 2 molecule associating with
3 invariant chains and forming this complex. And these, this, what invariant chain is,
is a some sort of chaperone and it, it gets the MHC class 1 molecule, class 2 molecule
together and then, and escorts this, them to a compartment, known as MHC class 2 compartments
or also known as M2C. And M2C is, is the way, usually by which, these are described and
these are lysosome like compartments. And so, once it, it goes in there and there invariant
chain is then degraded by cathepsins and other proteases.
So, once, so, the way it is, you have MHC class 2 molecules, they associate it with
invariant chain and as a deformer, trimer of trimers, and then this complex go to the
M2C.
And it is in the M2C, that invariant chain is degraded except for 1 part of it and that
is known as the CLIP and that is, the CLIP is the class 2 associated invariant chain
peptide. So, now, you have a complex known as MHC class
2 and associated, that is associated with CLIP and that is a, sort of, remnant of the
invariant chain. Now, how does one get rid of this, this peptide that is associated with
it? So, there is another molecule known as DM. Now, we had come across DM before. Now,
DM is encoded in the, in the, the MHC and it is the genes allocated in the place where
the LMPs and the TAPs are also located. Now, what DM does is, it catalyzes the removal
of CLIP and in fact, DM acts as an editor of MHC class 2 binding peptides.
So, what DM tries to ensure is that you have very high affinity binding peptides or slow
off-rate peptides, that binds MHC class 2 and so MHC class 2 molecule would be stable.
And I have I talk to you about the analogous role of tapassin in MHC class 1 that has a
similar sort of function. And so, once DM does the catalysis, so, so, if, so here is
a question, that I like to ask you, if in cells if there is no DM, what would happen?
What would happen is that you would have MHC class 2 molecules associated with CLIP because
there is no DM to take it off the, the CLIP molecule. The CLIP association with MHC class
2 is a not a very stable association and, and that is why, the MHC class 2 molecule,
that are formed over here are not very stable. So, what DM does is removes the CLIP of, and,
and gets, you know, the peptides, that bind with the slower off rate to MHC class 2 molecules
on. So, and then, once you have now properly well-formed MHC class 2 peptide complexes,
they sort of egress on and they are expressed on the cell surface. So, the way MHC class
1 molecules are formed and the way MHC class 2 molecules are formed, they are very different
and you should be a little bit aware even in terms of cell biology, the localizations
and all are very distinct, and that is a very important aspect and you should think about
that. So, what would happen is antigens, that are generated, you know, within the cytosols,
they are by, by and large, the peptides are generated near the MHC class 1 pathway.
Whereas, pathogens are entire VL isozomes or are endocytose, those, those peptides are
generated in M2C and they are found, there, there peptides are expressed by the MHC class
2 molecules. So, what from, from the host point of view or from the cell point of view,
it has taken care of both parts, both the endogenous as the exogenous pathway by which
proteins are processed and can enter cells.
So, a little, some differences between invariant chain and DM, which is known as, also known
as editor-in-chief. So, invariant chain is, you know, as the name suggests, is invariant
and DM, there are some polymorphic residues known; invariant chain is not linked to MHC,
this is MHC linked. Where is it and I think, what we will do is, I will, I would like to
just show this.
So, this is, this, this is the MHC class 2 region and the, these are the proteosome genes
and this is DM, this is where DM and this part is located between DP and DQ, who, and
over here, this part is located between the K and the MHC class 2 locus over here, and
you can see, this is where DM is and this is where the proteosomes and TAP genes are.
So, it is right in the cluster of those 7 genes, that, that were found.
So, the invariant chain, the job of the invariant chain is to take the complex of MHC class
2 molecules and then, chaperon them to the M2C, and it does so by certain targeting motifs
and these are dilusive motifs. DM, on the other hand, goes to the M2C by itself and
it targets via a tyrosine motif. Now, its job mainly is to remove CLIP from the MHC
class 2 molecules. You see, in invariant chain, once it goes there, its primarily job is done
and therefore, you know, and that is why, it is degraded, you have to get rid, the messenger
is degraded and the part of the messenger is left on and that part is removed by a DM.
The invariant chain, it prevents proteins, peptides to bind MHC class 2 in the ER and
you know, but the main job of invariant chain is to chaperone it to the M2C, the main job
of, of, the main job of an invariant chain is that. The main job of DM is that it is,
is the peptide loading capacity of it. So, what would be the phenotype of an invariant
chain knockout mice and DM knockout mice? So, in the case of invariant chain knockout
mice, the MHC class 2 molecules will be stuck in the ER because they would not know how
to go to the M2C in the absence of invariant chain. So, there would be no expression of,
of MHC class 2 on the surface. Now, if there is no expression of MHC class 2 on the surface
or very, very low expression, what would happen is, there would be no selection for CD4 positive
T cells. So, no MHC class 2, no CD4 positives; no MHC no T cells.
And the, but in case on DM, what would happen is MHC class 2 would get expressed, but this
would be MHC class 2, that is an expressing CLIP, which is an inefficient peptide, but
there would be, so there would be some selection for CD4 positive cells. So, you would have
MHC class 2 expression, even though it is not the type of MHC class 2, that you would
like, but you would have MHC class 2, but the peptides are known to be low infinity
peptides, not high infinity peptides. So, the MHC class 2 molecule are not the way,
that you would want them to, but you would have MHC class 2 on the surface and you would
also have selection of CD4 positives, but, but it would be an altered selection because
the proper right of a peptides are not their own MHC class 2. So, you should, students
should be able to think a little bit about the differences, about the functional roles
of this especially in view.
Now, to make matters somewhat more complex, you have MHC, you have what is known as DO.
Now, you have DM and DO and both, DM as well as DO, are MHC class 2 like proteins, but
they do not bind peptides, but they have other jobs. DM is, as mentioned, was, is some sort
of editor in this thing; DO, on the other hand, its role is controversial. But what
has been shown is that DO and DM are often associated with each other, but the problem
with DO is that it is not expressed, its express, its expression level is, is found only in
some type of cell. So, for, for DM action, you really do not,
you know, DO is not required because you have some cells where there is no DO and still,
the DM functions and MHC class 2 is expressed. But when DO is there, it modulates DM function.
So, and DO for, is expressed only B cells and thymic epithelial cells and so DO modulates
DM activity and therefore, effects the repertoire of peptides, that bind to MHC class 2. Now,
in the H2 DO lacking mice, the MHC 2 peptide repertoire is altered compared to wild TAP,
is not surprising; what is interesting is that you have an increase in number of CD4
positive single positive cells. So, you have much more single positive cell,
perhaps because of alteration in this in peptide repertoire, and may be, you know, in selection
of CD4 positive is somehow being effective. Now, in recent days, what has been found is
DO inhibits the entry of activated B cells and remember, it is expressed in B cells.
So, it inhibits the entry of activated B cells to enter germinal centers because germinal
centers, you have the T and B cells being activated, and that is where actually, the
initiation of the immune response takes place, the B cells become responsive to the T cell
cytokines, so on. So, DO, DO seems to be affecting that, that also.
Now, MHC class 1 peptides are generated by proteosomes. So, what are the enzymes, that
are, that play an important role for MHC for the generation of MHC class 2 peptides? It
turns out it is a family of enzymes belonging to the cathepsins. Now, cathepsins are belonging
to the papain family and over here, they are primarily cystein proteases, which means cystein
is in the active side except for 2 of the more famous ones, which is, cathepsins in
D and E are aspartyl proteases, but other than that, they are mainly cysteinyl proteases.
What is also known is cathepsins in D, E, F, I, S, they are broad specific endoproteases,
whereas cathepsins in B, H and Z are exopeptidase. Cathepsins are primarily found in lysosome
compartments and lysosome or, or MHC 2 compartments. Now, there, you know, for cell biologists
there is a difference between lysosomes and the M2C.
So, it appears the, the, the MHC class 2 are present in specialized molecules, which are
lysosome like, but not exactly lysosomes and that is why, this name has been given as M2C
because the certain markers, that people look for, there are some differences between the
markers between the, the, the true lysosomes and the M2C compartments are, but cathepsins
are, are present in both these compartments and they are important for the generation
of peptides. Because once you have the proteins over there, they need to be broken down and,
and cathepsins are primarily responsible for it.
Now, in this, cathepsin B and D are the most abundant proteases, but they do not play a
major role in cleaving invariant chain. Remember, it, invariant chain shepherds in over there
and then needs to be clean. Cathepsin S, on the other hand, plays an important role in
invariant chain degradation; it is present in B cells and dendritic cells; it is induced
by, by interferon-gamma IL-10, which is an immunosuppressive cytokine; it suppresses
cathepsin S, expression and lack of cathepsin attenuates several autoimmune disease model
in mice. Now, so why would this be? Because there is a co relation over here between MHC
class 2 expression and, and autoimmune diseases, it is something that will be discussed.
So, what would be happen is, if you do not have cathepsin S, it might, there might be
lesser expression MHC class 2, which might affect the, the autoimmune diseases.
Now, MHC class 2 peptides can also be generated by cathepsin L and cathepsin L can in fact,
it can substitute for some functions of cathepsioin S, is present in macrophages and cortical
thymic epithelial cells. Now, here mice that lack cathepsin L have 70 percent reduced numbers
of CD4 positive T cells in the thymus and periphery; they also shed, therefore.
So, clearly, over here you can see where cathepsin L seems to be playing, you know, you know,
a role in MHC class 2 and as a consequence of that it is affecting the selection of CD4
positive T cells. So, not surprisingly, cathepsin L lacking mice demonstrate reduced autoimmunity
and reduce number of CD4 positive cells because they are, sort of, linked and that, something
that we will see. Now, apart from cathepsins, other proteases are also involved, asparaginyl
endopeptidase is shown. It is a cysteine protease that cleaves after asparagine and is important
for sync of antigen, especially, for example tetanus toxoid, which is a very well studied
antigen.
So, we will just briefly describe the MHC class 2 processing pathway. You have extra
cellular antigens, that come in over here and so, they meet. The, the peptides are generated
in the M2C and what is shown over here is that the MHC class 2 molecules are synthesized,
they bind together with invariant chain and invariant, they form a trimer of trimers and
they, invariant chain shepherds them to M2C, like M2C compartments, where they meet these
extracellular antigens or proteins, and then these are cleaved by cathepsins and part of
the peptides are, are bound to. Over here, you will remember, that invariant chain is
cleaved and you have MHC 2 molecules along with CLIP and then DM, you know, removes the,
the CLIP and then helps the MHC class 2 molecules load on with higher infinity peptides and
then, these are expressed on the, on the cell surface.
Now, we had discussed this aspect about the link between MHC molecules and disease. Now,
defects in MHC gene expression lead to immune deficiencies, so and the reason for that is
MHC molecules are, quest, are required for both T cell development and activation. And
this is something that we have, we have studied in this part of the lecture. And some MHC
alleles are associated with susceptibility or resistance to autoimmune diseases.
Now, one important disease is known as a bare lymphocyte syndrome. So, there could be several
reasons for the bare lymphocyte syndrome. So, what, what this means is, bare lymphocytes
means, there are, you know, there are less number of lymphocytes. Now, what, what could
that be due to? So, for example, if you have defects in TAP expression, so be a less can,
can be due to mutation in several genes, one of them, say, there will be in, in TAP. So,
if you do not have TAP, you do not have MHC or in class 1 expression, or even if you have
some MHC class 1 expression, those MHC class 1 molecules are unstable and so, because you
have reduced number of MHC class 1 molecules, you have less selection in thymic selection,
you have less number of CD8 positive of T cells and these lead to immunodeficiency,
these are respiratory. So, you will have a respiratory and skin infection.
Now, remember, over here, in this case, you are lacking CD8 positive T cells, your CD4s
are there. Now, in some cases you have transcription factors where, which control MHC class 2 expressions.
So, for example, very well-known one is C2TA. Now, in, in, in these sort of animals or,
or humans, this is important for expression of molecules, that are important in the MHC
class 2 gene expression, so MHC class 2 molecules, invariant chain and DM, so on.
So, if you do not have this, what would happen is, there would be no MHC class 2 molecules
or very less reduced expression of MHC class 2 molecules. What this would do is, you would
have less number of CD4 positive cells. In the absence of CD4 positives, which are important
in helping macrophages, helping in B cell help, so on, it results in severe combined
immunodeficiency and C2TA is an example of a very important transcription factor that
is important in, in MHC class 2. And since MHC class 2 is so important, it results in
severe combined immunodeficiency and it is a very important bare lymphocyte syndrome.
Now, there are some HLA types that are associated with certain autoimmune diseases. So, for
example, particular HLA allele, which is the MHC class 1 molecule, HLA B27, there is a
very strong association with spondylities; so, very strong association with HLA B27 with
spondylities. In fact, the relative, the risk is, is fairly high and you can see some sort
of risk along with other autoimmune diseases. So, assemble multiple sclerosis with a DR2
and myasthenia gravis with DR3, there is, even the rate of risk here is not as high
as the striking one with HLA B27.
So, the other example of a very high risk is insulin dependent diabetes, where, and
where the insulin producing cells are, sort of, are destroyed by T cells and again, there
is a big association with a DR3 and DR4 MHC class 2 molecules. So, again, as I said, so
you know, having those MHC class 2 molecules, what, what happens is that the chances of
a particular autoimmune peptide being presented appears to be high. As a result of which,
it triggers off autoimmune CD4 positive cells, which, which kill off, you know, certain,
I mean, in this case, there are diabetes are producing, the pancreatic beta cells are being
targeted and they are being killed off, and as a result of which you have this, pretty
your, your, risk is, is high.
So, we will try and summarize what we had learnt in these classes. So, in MHC class
1 and class 2, the main important point is that the MHC class 1 pathway is the endogenous
path, which means, proteins, that are synthesized in the cytosol and peptides are generated
from these through proteosomes. Peptides are then translocated from the cytosol into the
ER by the transporters, TAP and then, you have this peptide loading complex, where you
have the heavy chain and which is bound to, made it to macroglobulin and then, come and
meets them, your tapassin, which helps all those. And you have other proteins like caldex
and calreticulin, forms a big complex and then, once you have proper peptide loading
on, as the confirmation change and MHC class 1 molecules are, now you know, moves to the
surface by vesicular trafficking. The MHC class 2 on the other hand, it samples antigens
through the exogenous pathway. And over here, the enzymes, that are important over here
are the cathepsins and what cathepsins would do is, you know, degrade these proteins; peptides
are generated. Now, MHC class 2 molecules. Coming to the
M2C, with the help of the invariant chain, once they come there, the invariant chain
is degraded, but you have a part of it attached on, which is CLIP and this CLIP is removed
with the help of DM. And you have DO, which modulates DM activity, but now, and DM helps
load on peptides with higher affinity, slow off rate on to the MHC class 2. Once this
MHC class 2 molecules are loaded with optimal peptide, this, sort of, move on to the surface.
So, one could ask questions, what happens if there is no DM, there is no invariant chain;
if there is no TAP; if there is no blended to macroglobulin, and these are aspects, that
students should be familiar with. Mutations in assembly factors are important and this
is especially true because you have molecules, you know, these molecules play an important
role in diseases. So, for example, bare lymphocytes syndrome,
you know, the primary reason for this are, you know, TAP deficiencies or C2TA. We also
discussed MHC link diseases. Now, there are certain MHC, that if you express certain MHC
molecules, your, there is a high chance of linking with a particular disease with HLA
B27. You know, there is a very high risk with ankylosing spondylities with, with particular
DR. There is a very high risk with insulin dependent diabetes because the pancreatic
beta cells are targeted and they are killed off.
Now, why is, why is this, what happens is, those particular MHC molecules, they express
or they have a tendency to express certain peptides and those peptides may, may initiate
a CD4 or CD8 response immune response. So, it is, it is, it is a relative, it is a, relative
risk is increased if you have some of these MHC molecules. So, MHC molecules are certainly
very important for initiation, both for T cell selection as well as T cell activation.
So, so, it is a very important process. So, it is, so, how they are, how they are synthesized,
what are the assembly factor, it is very important to understand their pathways and even now,
you know, there are better understanding of, this may help us understand the MHC class
1, class 2 pathways more efficiently. It may help us design better ways to boost up the
immune response.