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Monday,Ziad Al-IbrahimMHC725

Monday,

iad Al-Nasser

Ibrahim

MHC

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ImmunologyMonday, July 11th.By:- Ibrahim Miqdady

(A long unsure-about introduction/revision ahead! Don't judge upon it, the lecture is nice.Sit back, relax and play some Yanni's!).

We had been discussing so far the antigen recognition molecules, immunoglobulin, T-cellreceptors. And today we'll be talking about MHC.

We talked about antigens and antibodies and their nature, structure, diversity and wediscussed in details how we answered their question how did we get to the number 10^11different specificities of immunoglobulin, and also we had concentrated on when we talkedabout the specificity on the variable region, and the V region is the one that determinesthe specifity while the constant region is the one that determines the biological function.And going form that concept into the variable region which is formed of variable genes(joining genes of the light and joining genes of the heavy chains) and how those genes getrearranged and one of the them is going to be selected.

Also we talked about in the primary immune response how the first antibody is developedas a monomer of IgM and how a monomer of IgD is going to be present on the surface inorder to complete the function of the T-cell receptor. So the first antibody that's going todevelop is the IgM monomer, whether it was surface or it is going to be excreted itdepends on the part that is going to be activated; the one that goes to the cytoplasmicmembrane.

And the simple immune response here, that the other constant genes (gamma 1,2,3 ..alpha 1,2 .. epsilon) all are there. They have not been deleted. In the secondary immune

response there'll be the isotype switching or class switching where the variable regionstays the same, only in the development instead of selecting Mu or Delta, one of theothers will be selected and the rest will be deleted. So that particular cell will becommitted in the secondary immune response to produce antibody of the same specifityof the IgM and IgD, the difference here is in the constant region. And what really triggersthis switch into that area depends on the cytokines that are produced and how cytokinesare going to modify the cell and switch that particular areas. (e.g. To produce IgE antibodyinterluken 4 is required which comes from T-helper cell. )

I want you to remember that in the secondary immune response isotype switching is goingto take place to produce a new antibody for that antigen. So advanced functions are going

to take place in the secondary immune response. So remember when we talk aboutisotype switching we mean keeping the variable region which is very specific constant (notchanged), we only switch from (Mu,Delta) into others. And the switching here is by theaction of cytokines that are produced by the T-helper cells. The other constant genes aregoing to be deleted. That memory cell will keep producing only that particular antibodyisotype.

We also talked about the allelic execlusion And how these allotypic markers can beinherited one from each parent but one of them only is going to be expressed and theother is going to be deleted or non-functional so we don't see it. We talked about theidiotypic antibodies and the significance of the hypervariable region. And in comparisonwith the T-cell receptors where we have 2 types. TCRs are formed of two peptides (alpha

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and Beta) that are forming 95% of the TCR-1 and 5% of TCR-2 (gamma and delta). (notsure of what he said!).

And I told you the number of those genes responsible for those regions, they are so many,so the diversity is going to be much much higher. The difference between (Gamma Delta)and (alpha beta) is that the first is less specific; non-polypeptide antigens can bepresented (mainly on the skin or the mucus membranes rather than other tissues) .. andhere the difference is that we aren't going to have what we call ''somatic hypermutation";a

better fit.

We said that in the TCR requires that the antigen has to be presented with a MHC, whilethose others can bind without that MHC.

And finally about the molecules that are so important for the function of the T cell. Wetalked about the Cluster Designation/Differentiation Antigens (The CD system; 1,2,3,4) ;we consider them as cell markers, and each one of those have special function in thecontext of its presence (where it comes). For example when I say CD2, this is present onall lymphocytes (irrespective of the type of that lymphocyte I can target the CD2). CD3 isso important for the signal transduction and present on all T cells. CD 4 is present on T-

helper cells. We said how the CD4 plays a major role in the function of the T-helper andhow it has to match with Class 2 MHC . While CD8 (T-cytotoxic cells) has to match withclass 1 MHA.

You will see how the matching is so important for the signal to pass through into the cell,and also the lymphocyte functional antigen, the ICam-1 and so on. We said those areso important accessory molecules and if we are lacking any of those we are going to haveimmune compromise condition; diseases. Or if one of the enzyme we have talked about ismissing ; RAG1 or 2 that is very important for the gene rearrangement at the germ line ;the patient is going to be immune-deficient, and the diseases that are associated with

those.

TODAAAAAAAAY(yes, finally!)we will be talking in more details on the third part of theantigen recognition molecules. And those differ from antibodies and T-cell receptors in thatthey are not that specific compared to those two.

They are simply proteins that are coded by genes present on the short arm ofchromosome 6. Each of us have on chr6 areas of genes (called loci). Those genes aredivided into 3 classes; class 1,2 and 3.The ones that are involved in the immune system are simply class 1 and class 2. Class 3

might be involved in other things; enzymes, some complement components....

So we are talking about classes; we called them loci on the chromosome, and those areashave different allelic forms; variations of the same gene. And we inherit those allelic formsfrom our parents; half from each. So both are going to be expressed on the surface of thatcell.

It's important to differentiate this from the allelic exclusion. If the mother and the fatherare related to each other, then the chance that these variations will be less, and viceversa. Many allelic forms are present, each one of us will have two of these allelic forms,so the total number in type 1 MHC is 6 forms and minimum of 3. And in class 2 the samething, a minimum of 3 and a maximum of 6; with a total of 12 (Maximum) to 6(Minimum). And those form what we call a haplotype (It is a combination ofalleles at

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different places (loci) on the chromosome that are transmitted together); its like a stampfor each one of us, those antigens that are expressed we call them HLA antigens; ortransplantation antigens, or MHC antigens.

The major role of those HLA antigens is to regulate the immune response, its not that theirrole is just to reject grafts. Rejection of a graft is one of the mechanisms of our immunesystem; that those grafts are foreign so we don't want them to be there. If they are similarto our tissue then we want them to be there, or we can modify our immune system to

accept those tissues and so on.

Remember that all MHC antigens that we have on our tissues are the same, and those aredetermined by our genetic setup that we have inherited from our parents. If yours andmine are the same then we can exchange grafts, if different then we are going to rejectgrafts from each other. We are talking about genes on chromosomes.The Short arm of chromosome number 6 codes for proteins; Class 1 and Class 2 MHCgenes.

Class 1 codes for a single

polypeptide chain; alpha (45kD) attached to it a 12 kDBeta-2 microglobulin that hasnothing to do with thefunction of the alpha chain; itjust adds up support to theAlpha chain; it will make it doits function much better, andits coded by a differentchromosome rather than

chromosome 6.Any nucleated cell in our bodyis going to have class 1 MHCantigen. So RBCs do haveneither class 1 nor class 2, andthis makes them easy to betransplanted; we don't have tolook for MHC because they arenot expressed on the surfaceof the cell, but they arepresent on the surface of white

blood cells; because they arenucleated.

# So does our body produce antibodies against HLA antigens on white blood cells when wedo blood transfusion? Yes of course! But those are not going to annoy us, because whenwe do blood transfusion we are concerned about RBCs, we are not concerned with theWBC; those might sometimes cause a little of fever.

Class 1 is present on all nucleated cells of our body and class 2 is present on the surface ofthe antigen presenting cells (APCs), so the APCs are going to have class 1 and class 2,while others are only going to have class 1.

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Class 2 are formed of 2 polypeptide chains; alpha and beta. And those are going to bepresent on APCs of our body.The MHC molecules are concerned with antigen recognition, we will see that in each allelictype the shape of the alpha and the groove that they form has to do with antigenrecognition, so the antigen has to fit in the groove between alpha1 and alpha2 domains ,and this is going to be presented onto a T-cell, so heres what I called dual recognition; onerecognition on MHC; the group has to fit to the antigen, and the other side, you need the T-

cell receptor to go in into the MHC antigen and the antigen at the same time. So weretalking about antigen-recognition antigen, in humans we call it the HLA (Human LeukocyteAntigen), in Murines, mice and so on we call them H2, in dogs they have different name etc.

On the Short arm of chromosome number 6 we have areas called loci; three loci for class1 [ locus A, B and C] and other 3 for class 2 [DP, DQ, DR]; and these have extensivepolymorphism meaning that people are so diverse of the HLA antigen, so its so difficult tohave a match (i.e. tissue match) and here when we say polymorphism we mean manydifferent haplotypes; and this haplotype is what we need to identify when we do tissuetransplantation; identify the map (haplotype) for each the donor and the receptor, and if

they match then we accept the grafts, but if they dont, the grafts will be ultimatelyrejected.

This isthe short arm of chromosome number 6 here, this is the centromere over here (can yousee it? I can't. blah!) , this is how those genes are located, and in the farthest end we havelocus A then C then B , so remember B, C, A just going to the end here.

And then we have class 3, then class 2, this is how those are arranged. So class 1encoding molecule that present the antigen to the T cytotoxic cell (CD8+ T cells); this isthe main function of class 1 MHC, and it makes sense (Ah sure does!); class 1 MHC antigen

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is located in all nucleated cells, if these cells are tumor cells or virally infected cells thentheir antigen has to be presented with class 1 MHC antigen to the T cytotoxic cell, so the Tcytotoxic cell will refuse to do the killing unless the tumor or virally infected cell ispresented with class 1 MHC antigen.Locus A has many different allelic forms, maybe the highest number is for locus B ('maybe'he said!) , over 7oo or 1000,and you are going to have two of each, one from the fatherand one from the mother, remember that; for the B, for the C and for the A, I think the C isthe lowest, but the majority of allelic forms are present in B, and every year they discover

new allelic forms and they add them up.

Class 3 is able to encode complement or cytokine molecules, and some other enzymes forsteroid metabolism, proteins and so on.Then we have class 2, and as you can see class 2 MHC antigens are formed from twochains, alpha & beta, and as you can see the DR, DC, & DQ); (check the figure above), thisis how they are arranged. And in between in class 2 we have areas that will code forproteins that are needed for the processing of the MHC antigens like the low molecularweight protein LMP, and the transporter associated protein TAP. Those (LMP andTA) are needed genes involved in antigen processing; they process the antigen, and thenthey will be mixed with the MHC, and then it will be presented to the T helper cell. This is

all present in the short arm of chromosome 6.(Remember thatin Class I MHC antigens in locus A, B, & C, in each of these there are twoallelic forms; one from the father and one from the mother.)

These are the different allelic forms that weinherit from our parents on each locus;locus A has 414 different allelic forms, wehave two of those on the surface of ourcells. On locus B we have over 700 (around1000 of those have been discovered). Locus

C is the lowest ; 210 different allelic forms.And then we talk about Class two(DP,DQ,DR); alpha and beta. And you cansee how many allelic forms and thevariations that we're going to get. The DR-beta is the highest form (over 500 differentallelic forms).

The MHC genes are extensivelypolymorphic. We have so many of those inthe human population; existence of

multiple alleles at a locus; 2 at each locusfor one person. HLA-B and DR-beta are themost polymorphic loci shown here.

This inheritance is a comdomionance typeof inheritance, and you can see here thechromosomes (diploid) . (The child isdifferent from the father and from themother)

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For example if I want to do a transplantation from child (1) to child (2) , actually you aregoing to introduce something foreign into the other child, while for example if we introducefrom the mother or the father into the child then this is accepted much better in a away,because here the amount of tissue that will be added at least matches half of the genesthat the mother or father have. None of family members have identical combinations ofHLA alleles and thats the main reason why tissue transplantation is difficult. The easiestway for transplantation is to have a match between haplotypes and the best place to havea 100% match is to use a tissue from one place from our body to another. This is done in

birds.

Autologous; taking a tissue from one place in your body and transplant it onanother place, for example in our hospital we do autologous bone marrowtransplantation; we take the bone marrow of the patient out and then you exposethe patient to radiotherapy, cytotoxic drugs, or make suppression. Then the bonemarrow is transferred back to the body. So autologous is the one that have almost100% match with almost no reactions at all.

Syngeneic; in the case of identical twins. The haplotypes are the same in thiscase. So we can take grafts without any problem.

Allogeneic ; You have to do

the haplotype for the donor andthe recipient to get the bestmatch. Its impossible to find a100% match but some choices arebetter than others (e.g: parent tochild, brothers and sisters, 1stcousins then 2nd cousins and soon). Those are genetically diverse.

Xengeneic. Animals areused and the pig is the closest

animal genetically to human. Theimmune system of pigs is almost

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identical to immune system of humans. So insulin body samples and heart valveswere used in the past, which were taken from pigs. Now they are trying to have totransplant pigs heart into human, but to do that they have to make the patientseverely immune suppressed in order to accept the grafts.

This is the expression of the genes when you inherit them. You can see the haplotypes; forthe father the green and blue (no colors? You need to have a wide imagination!), and for

the mother the pink and the orange. And the child has one from the father and one fromthe mother and both will appear on the surface. So there will be diversity and the childrenwont have identical haplotypes as those of the parents.

( the figure above)So when we look at the structure of class 1 MHC;-> Class 1 is composed of:

1) a single polypeptide chain (alpha chain); which has 3 domains alpha 1,alpha 2 and alpha 3 and its about 45,000 daltons. Which are external.2) Another part that goes inside the endoplasmic membrane.3) And theres another protein thats called beta 2 microglobulin which isaround 12,000 dalton and is adherent to alpha chain. This protein is encodedon chromosome number 15.

4) The sensitive part thats going to bind is between alpha 1 and alpha 2domains.

After the intake of the antigen by the cell it will be processed and presented to the surfacewith class 1 MHC antigen. So we need to present that to the T cytotoxic cell that has CD8on its surface.

(The doctor started to give further explanation about the same figure)

> In a 3D structure youll see that alpha 1 and alpha 2 represents a groove , if you havemore grooves here (or polymorphism) then the possibility of helping in the immune systemis higher.

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For example; the presence of 6 grooves is better and stronger than 3. Here we are talkingabout the shape of the groove; alpha1, alpha 2, alpha 3 and so on. You can see the beta 2microglobulin and that the beta pleated sheat is going to fit into that. Many of thepolymorphic amino acids in MHC class 1 are found here. This molecule structure doesntinclude the membrane standing region of the alpha chain. Beta 2 microglobulin is theprotein required for folding and expression of class 1 molecules. The alpha 3 domains andthe Beta 2 microglobulin pair to form a region of a very similar immunoglobulin domain. Sothe structure of that belong to the immunoglobulin supergene family. You can see domains

like these in immunoglobulins, variable region and a constant region (very similar toimmunoglobulin ).

( figure above)Class 2 :There are 2 polypeptide chains alpha (1 &2) and beta (1&2). The sensitive part here isbetween alpha 1 and beta 1 which will fix into the antigen. This is on the surface of antigenpresenting cell (APC); (e.g. macrophage, dendretic cell or B cell). These APCpresent the antigen to T helper cell which must have a T cell receptor that fits the antigenand CD4 and this CD4 antigen must match that of class 2 MHC antigen. This is the mostspecific part forming the groove between alpha 1 and beta 1.

The regulation of the gene expression of class 1 MHC antigens is on locus A, locus B, locusC antigens. And of class 2 MHC antigens is on DP, DQ, DR.

Characteristics of gene expression of MHC antigens:o Co-dominant mode of inheritance; half from the father and half from the

mother.o They are very similar to Immunoglobulins and TCRs in their specificity, so we

have dual recognition; which means that the antigen must fit in the groove of MHCantigen and then to be presented on T cell .

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o Theres no Allelic exclusion. Unlike Immunoglobulins and TCRs. So both genesare going to be expressed on the surface.

S tructures of MHC Ags:

Class 1 alpha chain (around 45 kD) is attached to B2 globulin that comes from other

chromosome, allelic forms have more than 90% similarity (e.g. between A1 & A2) sothe specificity difference between the alleles is about 10% , this 10% is important

but its minor . (This is different than in the antibodies and the TCR allelic forms), the

resulting groove will be shallow in a way so that it will accept many antigenic

determinants.

No gene rearrangements is going to take place, both is going to be expressed.

Precise sequence vs. amino acid residues; here the precise sequence is very

important in the immune recognition and response.

Class II : consist of two polypeptide chains (33 kD ) and (29 kD) chains, in

addition to the trans-membrane glycoproteins.

MHC molecule can accommodate a wide range of peptides (one at a time); as long

as you have 90% similarity, a wide range of peptides can fit in that groove.

Antigen recognition restriction

The antigen has to be presented with a self MHC in order to bind with the TCR, so

the antigen-MHC complex has to fit in the receptor of the T-cell, meaning the T-cell

is specific for both of them (the antigen and the MHC together form a new shape to

fit in the t-cell receptor) and this is called restriction rule.

MHC must be self and this is the dual recognition of the T-cell, if the MHC was non-

self , the immune response will not take place. The selection of the self MHC to bind

with the antigen takes place in the thymus gland, so the thymus recognizes the self

MHC and the strength of binding so that the self MHC will be released in the

circulation and the modified or the non-self MHC will be deleted or become anergic,and any problem in this process will lead to the development of an autoimmune

phenomena.

The MHC has a binding site for non-self antigen, the antigen has to be non-self

(foreign) and together they form a ligand for T-cell receptor . So the formula is

simple: non-self antigen binds with a self MHC both binds with the TCR.

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This picture emphasizes the idea of the dual

recognition. The one on the left is the normal

case where the non-self antigen binds with the

self MHC and together fit in the TCR and this is

the only case where the t-cell will be activatedto do its killing functions. In the middle, the

MHC is non-self so the complex is not going to

fit in the TCR and on the right , the antigen

does not match the specificity of the self MHC,

also no immune response is going to take

place

( .. continue Antigen recognition restriction)

Altered peptide ligand used for treatment : the antigen that is going to fit in

the TCR has to have all the characteristics mentioned above in order for the immune

response to take place , sometimes If I dont want the immune response to takeplace , I could modify the antigen so it wont have a perfect fit , this altered antigen

can be used as a vaccine in order to suppress the immune system (suppression of

the immune system is needed when the patient is suffering from an autoimmune

disease like multiple sclerosis and others we ). If I know the antigen that is going to

bind with the lymphocyte and I modified it so it doesnt have a perfect fit , its going

to give a negative signal and the lymphocytes will not be stimulated (immune

suppression).

This is the normal immune response

having the target cell, MHC, antigen

and TCR. If magnified the picture, you

can see the yellow antigen binding to

specific areas of the green class I MHC

and both the antigen and the MHC bind

to the TCR in specific places as well.

(No need for imagination here, it's

pretty clear without the colors!). So if

you altered the antigen in a way it will

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give a negative signal that will not let the T-cell to be activated, release its cytokines

or to act against the antigen. As you can see amino acids side chains from the

peptide antigen interact with MHC and TCR and the MHC interacts with peptide

antigen (anchor residues) and the TCR.

Also, they have noticed that certain autoimmune diseases have association with certain

HLA alleles; this is called relative risk: meaning that having a certain HLA may increase the

risk of having this specific autoimmune disease

For example:- Both Positive and Negative P27 people can develop Ankylosing Spondylitis but peoplewho have HLA P27 (positive) are at higher risk (90 times) of developing AnkylosingSpondylitis than those who dont have P27 (negative). P27 is a risk factor of AnkylosingSpondylitis. Its like saying smokers are at higher risk (120 times) of developing lungcancer, some smokers develop cancer and others dont but they all at a higher risk ofdeveloping it. Ankolosing spondalitis is an autoimmune disease where antibodies attackintervertebral disks and cause a problem with bending of vertebral column. We use theterm bamboo spine to describe this because their vertebral column is not flexible justlike bamboo. It seems that p27 allelic form patients have a modification in a way withother factors that will let their immune system to develop antibodies more in thosepatients compared to others.

- In Insulin-dependent diabetes mellitus where PQ2 allelic form are at higher risk of it. TheIg will attack the beta-langerhan cells in pancreas. Why do they do so ? Some say

Coxsackie B virus could attack and then the immunoglobulins for Coxsackie B virus willcross react with beta-langerhan cells, or sometimes other infections will cross react withbeta-langerhan cells, complement will be activated and cells will disappear. PQ2 positiveare at higher risk compared to PQ2 negative people.- In Multiple sclerosis PR2 allelic form are at higher risk compared to PR2 negative, myelinsheath will be attacked by our immune system.- Acne vulgaris is a skin disease where skin will be attacked.- In RA the synovial membrane of joints will be attacked by our immune system. And SLEwhere anti-DNA Ig against DNA and so the cells they will be broken and complementactivated and PMN will be called into the area and damage could be occur systematically.

CH.9: Review of Ag recognition

This chapter is a review of the previous threechapters.We talked about: Ig, TCR, MHC all are antigenrecognition molecules which means they all arespecific and members of Ig family and itsmembers all have Ig domain.

-MHC:polymorphism/diversity, co-dominantexpression.

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-TCR: recombination/diversity.

- Ig: recombination/diversity, somatic hypermutation.

In the figure to the right you can see:

- Ig : made of 4 polypeptide chains; 2 light, 2heavy.

-TCR: alpha, beta, gamma and delta. You cansee a specific part between alpha and beta.

- MHC class 1 and 2.

- KIR we will talk about it later.

- CD4, CD8 and ICAM.

All these proteins belong to Ig supergenefamily.

(Now dr.Ziad just read the tables from the remaining slides PLEASE go and read them.

And remember in Table 1: You should know where D gene exisst: in Igs heavy chain andin TCRs beta and delta gene; that's what he said).

~ That's it for today, thanks for enjoying the lecture, I know

# Yousef OdeibatI would like to do what you demanded but; putting your pic over hereis not really a good thing to do, no need for more followers you know :D Thanks my great

friend for your great help # Oday Younes I appreciate the effort your artistic fingers have done for me, keep up thegood music my friend