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Antibody Diversity (cont.)and the T-Cell Receptor

Mai Mazin and Sara Zuriqat

Ziad Al-Nasser

Sunday, 10/7/2011

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Immunology - Lecture 6 / Sunday, 10-07-2011Done by: Mai Mazin & Sara Zuriqat

"This lecture covers the remainder of Ch.6 and all Ch.7"

Anti-body Diversity (Cont)

We will continue answering the question of how we are going to get around 10 11

different specificities of immunoglobulins and around 10 16-1018 differentspecificities of the T cell receptors. We said that to answer this question weshould know the structure of the immunoglobulin.

The structure is formed by light chains and heavy chains, and those have avariable region and a constant region. The variable region is coded by differentgenes; the V genes and the J genes for the light chain, and the V, D, J for theheavy chain. Those genes are located on different chromosomes; chromosome #2for the kappa chain, chromosome #22 for the lambda chain, and chromosome #14for the heavy chain.

And we said that a gene rearrangement is going to take place during B celldevelopment -where B cell is the one responsible for making antibodies-. Whenwe talk about the gene rearrangement at the germ line, we will see that the V andthe J start joining each other and we need enzymes to be responsible for this; wecall them the Recombinases or the gene rearrangement genes; RAG1and RAG2.

Those are so important; that is if they are missing in our body then no gene

rearrangement is going to take place, and the patient is going to be severelyimmune compromised. We call it "severe combined immune deficiency" if thoseenzymes are missing.

When the genes are rearranged and become closer to each other, transcription isgoing to take place. After the transcription process, looping out of the introns orwhat we call it gene splicing is going to take place (we know we have exons andintrons in the primary RNA), and then they will come together starting with the L(the leading sequence) and then the genes from the 5' end to the 3' end. Of

course you know that the mRNA is almost a complementary copy of the DNA.

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So the gene splicing is going to take place; the introns are going to be spliced outwhile the exons come all together. Those exons go into the translation, so whathave been selected is going to be here expressed as polypeptide chain. Thispolypeptide chain will be either a light chain (either kappa or lambda )* or a

heavy chain.

*This depends on which one is going to be selected randomly of course. The samething is going to be applied on the heavy chain; which heavy chain is going to beselected first.

I said previously that the and are going to be selected first; because they arethe ones to be shown as B cell receptors. And then after the IgM & the IgD havebeen developed on the surface, we have what's called excreted IgM. And whenthe B cell goes into secondary immune response the same cell (memory cell) willswitch; meaning that it'll keep the variable region intact and change the heavychain; so instead of having IgM, we are going to have for example IgG1 (if theswitch selected the 1) with the same specificity of the chain. This is calledisotype switching .

So the antibody diversity depends on how many of those genes (the V, the J andthe D) we are having, and then the recombination that is going to take place, andthen after that gene splicing, and sometimes what we call junctional diversity thatcould take place.

Junctional diversity: in the variable region the last 100 genes coded for the last100 amino acids of the light chain. Those genes could be added or deleted at thisarea and we call that junctional diversity. The enzymes needed for that called TdT (Terminal deoxynucleotidyl transferase) that will add nucleotide into that area soit will add to the diversity as long as it is in the variable region.

In the secondary immune response we have what's called somatic hyper-mutation where here a mutation is going to take place to give you what we call abetter fit.

This is (slide #4) a summary of what we are going to talk about:

1011comes from the calculation of (how many V genes we have) x (howmany J genes we have) this is for the light chain x (how many V of theheavy chain) x (how many of the D) don't know why the doctor droppedthe J genes of the heavy chain. We add into that the somatichypermutation and the junctional diversity and the chance of which lightchain is going to bind to which heavy chain.

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We are dealing with heavy and light chains; variable and constant regions.The variable region is the one responsible for the specificity while theconstant region deals with the biological function.

Coded by different gene segments.

Rearrangement during development; if you don't have it, you'll end upwith immune suppression. It starts with a leading sequence, j, and D genesin addition to V and C.

Many to select from, brought next to each other, the gene rearrangementand the enzyme Recombinases.

Somatic recombination, products then they assemble. Remember V for binding, C for biological function.

Referring to the figure; presents on Ch.2, this is the DNA at the germ line. Youcan see how the genes are arranged and one from each is going to be selected.We have 35 variable genes of the (the last 100 AA are coded by them). If weselect one of those V genes in addition to one of the 5J (J1-J5) plus the C regionwe'll end up with the light chain. Its specificity depends on whether we selectV1, V2, etc

*So for light chain one of the V, J should be selected plus the C k..

The is the same thing, the difference is just that is on Ch.22 and here you havemore than one constant region C 1, C 2, So you have 4 different types of and this is for biological function (has nothing to do with specificity). The onewhich has to do with specificity is the variable; about 30 V and around 4 or 5 J.

In the heavy chain we have more genes so more variation; around 50 V genes andwe have about 25 diversity genes (D) and around 6 J; those they make thevariable region of the heavy chain. The D genes are the difference between the L

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& the H chains & then we have the constant region. when we are talking about itwe are talking about the isotypes and the subtypes; the classes and thesubclasses; , , 1, 2 . One of them is going to be selected tomake that particular constant region of the H chain.

*In the light chain; V & J

*In the heavy chain; start with D&J then the V >> VDJ

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

We start with the V, J at the germline> VJ loint by somatic recombination and DNArearrangement> transcription to RNA the same VJ> then the J-C intersegment inblue is going to be spliced out> VJC> protein synthesis. Could be kappa or lambda.

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*however any given Ig molecule should contain 2 identical L chains.

Diversity also will be enhanced by a mutation that could be taking place at the

junctional phase between the J and the V. We call that junctional diversity wheresome nucleotides will be added while some will be deleted (addition/deletion typemutation) the enzyme required here is TdT.

So if you look here (the table below) to the initial gene segments, and what theyare going to code for, we can see that a nucleotide deletion causes the Aspargineto be replaced by Isoleucine and this will give you an additional diversity. Thesame with TdT; Glu will appear and it'll add also to the diversity.

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

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Now look here on the B cell

How IgM & IgD are going to be added to the surface of the cell?

The most important thing is that IgM & IgD must have the same specificity andthat makes sense, and the change is going to be only in the constant region. Youcan see here we have VDJ in a heavy chain of course. Then we have like a switchregion we call it polyadenylation A1 after C . Then we have the constant delta andwe have PA2. So it depends on whether PA1or PA2 is going to be stimulated atthe mRNA (what is going to be stimulated is going to be expressed). This is calledalternative splicing ; alternative means one at a time:

Either PA1>> C >> IgM

Or PA2 >> C >>IgD

And both of them are going to be expressed on the surface, so the variable regionis the same in both & . The difference in the C will have different functions.

What really decides for IgM to be secreted and stay on the surface of the B cell as

a B cell receptor also determined by thegene expression.

If you look carefully, this is the surfaceIgM. It has this green part of 2 parts, oneon the cytoplasmic membrane & oneinside the cell. Those (the green ones)have to be coded by genes, while theothers as you can see they are the same.

So the difference on thistransmembrane section that's if that is

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selected (T m1 & Tm2 by stimulating PA m) it's going to be a surface, if it's not thenit's going to be secreted.

So if you look to here; the variable region is the same> same specificity, we havethen C the surface part (pink) & the membranous part (green); T m1 & Tm2 havepolyadenylation part. It depends on which is going to be stimulated if PA m isstimulated, the green part will be added, and will determine that the resultantprotein is going to function as a part that will hold the Ig into the surface & theIgM will act as a B cell receptor.

So B cell receptor definition: it's a monomer of IgM, the only difference betweenthis & the secreted form is the transmembrane part that's going to be coded bygenes, and we have the polyadenylation sites that's going to be stimulated tocode for that particular part (the green one).

This slide shows the rearrangement part & how we are going to detect that bymolecular microbiology techniques.

We get here a DNA from fibroblasts, B cancerous cells, and polyclonal cells andyou can see how those genes have been rearranged at the malignant sidecompared to others that they don't fixed.

They say that these types of experimentations are nowadays old & can bereplaced with polymerase chain reaction to follow up the J, V or the D from onelocation into the other.

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This is what we call allelic exclusion . Do you remember when we talked aboutisotypes, allotypes, and idiotypes? We said that isotypes are the antigens thatform the heavy chain and the constant regions, they are the same within the samespecies; so all of us we share the same isotypes. And when we have otherantigens that could differ we call them the allotypes, so they are added up intothe heavy chain we call them marker 1, marker 2 , etc. For example, IgG1 (m 1) ifwe have marker 1.

If you have marker 1 and I inject mine into yours nothing would happen, but if youdon't have it & I do have it then you are going to respond to this marker.

Those markers are inherited in a process we call it allelic exclusion; one allele is

going to be expressed & the other will not. So if you inherit m1 & m2 only one ofthem is going to be expressed (not exactly the same as MHC where we have co-dominance; part from the mother & part from the father). So this is allelicexclusion; one is going to be excluded & only one is going to be expressed.

So if you look above at the parents with these markers in red or blue, each onehas a marker of its own, so when you look to the inheritance of allotype1 & 2 onlyone is going to be expressed on the surface either the red or the blue. No blueand red on the same cell as those of MHC antigens. When we are talking aboutallotypes we talk about them as we talk about the type of inheritance of theblood groups exactly.

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*Remember allelic exclusion> exclude one allele on the surface of a particular cell.

Chapter 7: The T- Cell Receptor

What about the T-cell?

The T-cell exactly follows the same format as those of immunoglobulins. So when

we talk about the T-cell receptor (TCR), how does it differ from that ofimmunoglobulins and what are the similarities between TCRs and IGs?

Summary of Ch.6:

To make a specific anti body (AB with certain specificity) youhave to:

1. Build a light chain either: Kappa by select 1 V gene out of 35 & 1 J gene out of 5 plus

the constant Or lambda by 1 V gene out of 30 & 1 J gene out of 4 plus

the constant (of 4 types)2. Build a heavy chain by select 1 V gene out of 50, 1 D gene

out of 25 & 1 J gene out of 6 plus the constant (ofdifferent types; 5 isotypes; M,D,E,G,A)

Bearing in mind that changing the C without changing the Vwill not contribute to the specificity only to the biologicalfunction.

The previous plus the somatic hypermutation and the junctional diversity and the chance of which light chainis going to bind to which heavy chain all contribute tothe Ig diversity & having about 10 11different specificitiesof Ig.

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**Differences between TCRs & Igs:

1) Immunoglobulins can bind to antigens freely and form immunocomplexes

while TCRs cannot do that. In TCRs the antigen has to be presented with aclass 1 or 2 MHC to the T cell receptor.

2) The TCR does not have what we call "somatic hypermutation" (a better fitin the secondary immune response).

Also from Hayat archive

3) We have so many joining (J) genes that are associated with the TCR ratherthan Igs. So, this will add up into the diversity, also you have Junctionaldiversity.

4) The TCR is found in two polypeptide chains while in Igs we have four .

**Similarities between TCRs & Igs:

1) The outcome shape of both of them which produced by theimmunolglobulin super gene family is very similar.

2) The TCR goes almost through the same changes that occur with the Igsdiversity.

So

here we are talking about germline TCR genes which will form the two

polypeptide chains and for TCR 1 that form 95% of TCRs, and the twopolypeptide chains and for TCR 2 which found in a little percentage. So wehave germline genes and , then gene rearrangement, the first one will act as alight chain and the second one will act as a heavy chain. The second one; the chain will have the diversity (D) genes, both of them will have J genes, then wehave transcription and translation then we will have and polypeptide chainsthen have a TCR assembly.

As a summary:

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1)You have , then 2)you have the V(D)J, recombination, transcription,translation and then this will code for the or polypeptide chains and 3)thenthe TCR assembly takes place.

In TCRs the number of genes that we are dealing with is much much morecompared to those of Igs. So we expect the diversity to be higher; around 10 ^16 10^18different specificities of the TCR. So it makes sense that we need to havemore specificities of T cells because of the nature and function of these cells thatthey are going to help T cells as well as B cells.

So if you look to the structure of TCR (below) you can see two polypeptide chainsand , while in immunoglobulin we have two heavy and two light chains (4

chains as total).

The specific part here -which we call it the variable region of the and - is theupper part which is exactly like the variable region of the heavy and light chains ofIgs. Also we have to notice that the antigen that will fit to the specific region ofTCR must not be free; it has to be carried on MHC molecule.

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

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Here we are talking about variable and constant regions the same as Igs. So whatis the difference here is that we have variable and constant for the chainalso variable and constant . So the most specific part of this TCR is going to bethe area between the V and V which is a relatively flat antigen recognitionregion that interacts with peptide antigen and MHC antigen and this is thedifference between TCR and Ig.

This structure is for the extracellular portion ofTCR. So we have an extracellular portion and anintracellular portion and here we are concernedwith the extracellular part. This is the mostvariable region (the flat surface in the figure) thatwe want the antigen and MHC antigen to fit into.So the diversity is going to be the same; that ithas to be applied to the chain that representsthe light chain and the chain which representsthe heavy chain, and this is for TCR 1 while for TCR2 represents the light chain and the heavyone. So the difference here is that the chain isgoing to have the D (diversity) genes and chainis going to have it as well.

So look here (at figure 7.3) these are the and chains which will form the TCR1.The will act as the light chain and it has around 80 different V genes and around50 J genes, so you see how much diversity we will have while in Igs there are 5-6genes (probably of J genes) as a maximum. While for chain there are violet color Dgenes, we have here two of them but they are variable in number; so they couldbe more than that. Also there are around 50 V genes, 6 J genes and constantregions.

Figure 7.3

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I have just said that this is the TCR, and it does not accept the antigen which is inyellow freely; the antigen has to be presented with class 2 MHC antigen on theantigen presenting cell. If you remember when we talked about the antigenrepresenting cells, we mentioned the thymus dependent antigens, macrophages,dendritic cells, inter vegetative cells as well as B cells.

And in all of these cells the antigen has to be taken in, processed and then mixedwith MHC antigen based on the specificity. As you see in the figure, this is thespecificity that should be applied with the antigen. Then TCR should bind both

MHC antigen (blue in color) and the antigen (yellow in color) which binds to it;that the antigen when bind To MHC antigen they will form a new shape that hasto fit in TCR and we call this restriction which means the antigen has to bepresented with an MHC antigen to the TCR and TCR should fit both of theseantigens.

The complex of MHC antigen -which binds a peptide antigen- is the ligand for TCR.And the top of the MHC molecule and the peptide form a relatively flat surface asyou can see in the figure. Also you can notice that only some residues of thepeptide antigen interact with the MHC molecule; so a part interacts with TCR asspecificity and part interacts with MHC molecule as a total. And I will keeprepeating all these information so frequently because we need it when we talkabout T cell activation and what really triggers the T cell to be activated.

Slide 23:

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Also here when we talk about specificity, this is the rule of the thumb * that wealways talk about in the thymus dependent antigens (adaptive immune response)but sometimes some exceptions could take place: if you look to the combinationin the figure in the previous slide, you can see the flat surface of TCR binds to MHCantigen and peptide antigen as I told you.

But sometimes some antigens we call them superantigens bind to the beta chainwithout having anything to do with the specificity of TCR or MHC antigen, theyjust bind to the beta chain in order to give a signal to the TCR then TCR is going tobe activated.

* So the rule of thumb is to have to be in combination with MHC antigens to induceresponse, the exception is the superantigens.

We see that in some of the toxins produced by bacteria, such as Toxic ShockSyndrome toxin that is produced by some strains of staphylococcus aureus(group A beta hemolytic strep). The toxin is absorbed through the vagina in thosewho use tampons (absorptive applications for the menstrual cycle), through that

it will be absorbed directly to the blood and it will interact with T helper cells inthis way (binding to the beta chain).

So as a result, T helper cells will be stimulated and start to produce cytokines(suchas TNF) immediately which lead to increase the temperature of the body andcause tissue destruction. Also there is another example of these toxins which isErythrogenic Toxins; the cause of scarlet fever.

"Superantigen can bind directly to the beta chain of TCR without binding of MHCantigen to the peptide antigen. So these antigens are not specific, which meansthey do not have to fit into the specific part of the TCR, just when thesesuperantigens bind to the beta chain and between the MHC and the T cell is goingto be activated. Also you can notice from the figure that the MHC binding doesnot involve the peptide groove".

Slide. 24:

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Here you can see a presentation of class 1 and class 2 MHC. Class 1 MHC consists of3 domains 1, 2 and 3. Another one which is for support called 2 microglobulin. Also you can see the specific part between 1and 2 which has tofit to the TCR.

Slide 25:

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

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We start describing the antigens which present on B cells or T cells by using theCD system. CD we call itcluster of designation or cluster of differentiation antigens and each antigen we give it a number (1,2,3,), then each one of thoseCD1,CD2 ,CD3,. has a function and we have to know the function of each one as

time comes to describe that particular function.

CDs (CD1, CD2,) are detected by the Flow Cytometry and we call themsometimes tumor markers if they are present in a mature cell where they shouldnot be present and vice versa; in an immature cell and so on. You will know moreabout this when we will talk about tumor immunology.

Refer to the figure:

This is the TCR ( and or and ), and this is an accessory molecule we call it CD3which is a complex of 4 polypeptide chain -sorry we didn't get what the doctor saidabout the structure of it but this is what is written in the book "it's a complexcomprising 4 different transmembrane protein chains "-we have two zetaand we call it zeta zeta*.

The function of the CD3 is to let the signal pass through. So when the antigen

binds to MHC then the CD3 lets the signal pass through, and as a result, there willbe activation of a biochemical pathway that will let some changes occur. So CD3has a very important role in letting the signal pass through and if it is not foundnothing is going to happen.

*from the net; it seems that also there are 2 chains.

We also have other types of accessory molecules which called CD4 and CD8. CD4

is present on the surface of T helper cell mainly but could be presented on somemacrophages and antigen presenting cells. And we will see how CD4 is alsoimportant for the signal transduction and it recognizes class 1 MHC antigens.

The same thing applied for the CD8 which presents only on the surface of Tcytotoxic cells and it only recognizes class 2 MHC antigens in order to let thesignal pass through, and then activates the T cytotoxic cell.

We have other accessory molecules such as the CD11A and the LymphocyteFunctional Antigens which play a major role as co-receptor molecules byenhancing the signal passage and activation of the T cells.

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The End

Sub7an Allah, such a perfect organized system working on.. So don't worry; yourbody is in safety! What's left is your soul, try to protect it

Done by: Sara Zuriqat & Mai Mazin