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Wednesday, 13/7/2011

Ziad Al-Nasser

Saad Aldeen Mohammed

Lymphocyte activation

and hematopoiesis

9

30

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The Doctor said that we gonna change seat numbers after the first exam

which is on Saturday 23/7/2011. And it will be at 8:15 AM.

Yesterday we were talking about antigen presentation , the role of MHC

antigens 1 and 2 in the presentation process ,the antigen presenting cells

"APCs" (macrophages, B-cells ,dendritic cells) ,MHC restriction phenomena ,

and we said that : what really determines whether class 1 or 2 MHC will be

involved ,whether T-helper or T- cytotoxic cells will be involved, is by the

way or mode of trafficking of the antigen through the APC .

And we said that we have 2 pathways:

1-Intracellular.

2- Extracellular:which could be: endocytosis(which involves toxins and

vaccines) or phagocytosis (which involves bacteria) . And we said that the

outcome of that is the production of antibodies in the case of endocytosis

or activating macrophages to get rid of intracellular bacteria such as

"mycobacterium tuberculosis".

And we said how antigen when taken in, it will be processed and cut by

proteases into small peptides ,and how it will bind to class 2 which is

produced in the endoplasmic reticulum (ER).

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And also we said how the class 2 will be covered by the invariant chain and

then it will pass though the Golgi apparatus ,and how the antigen will bind

to the MHC 2 in the acidic vesicle and then it will be expressed on the

plasma membrane so the T-helper cells that has T-cell receptor and CD4

which is really very important can come and bind with its ligand (the

antigen +MHC 2).

And then we talked about the other pathway which is the cytosolic where it

involves the intracellular viruses, tumors as well where we require

cytotoxic T cells to kill the virally infected cells or tumor cell.

So the virus will be processed and cut to small pieces and these pieces

antigen have to be passed through the ER where MHC 1 antigens are

located by transporter enzymes (ex:- TAP1 and TAP 2 ).

Then it will bind to MHC 1 antigens and transported to be expressed on the

plasma membrane to be recognized by CD8 cells .

We have talked also about evasive organisms like herpes viruses, adeno

viruses which can manipulate these MHC 1 molecules so they are successful

pathogens.

We talked about DNA vaccines and so when we need to develop acquired

immune response and memory cells related to T- cytotoxic cells we

introduce the DNA of the organism and it will be processed by the

intracellular pathway (as if it has developed within the cell) and T-cytotoxic

cells will be activated .

And we said that this is the major arm for the activation of T-helper and T-

cytotoxic cells by MHC restriction but we have other important factors we

will talk about which are the cytokines which will be produced by the APCs.

Then we started talking about lymphocyte activation, and we said that we

have to understand the biological and biochemical events that will take

place in the activation process to understand why immune suppression

could take place, how can we induce an immune suppression, mechanisms

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of action of immune suppressive drugs that we need for successful

transplantation.

Transplantation will not be successful until we suppress the immune

system.

So we said that the activation of T or B cell requires a stimulus, which is the

binding of antigenic determinant with the receptor on the cell. It's not just

binding, its a cross linking that will take place which is very important.

So in this antigen, as long as binding and cross linking are taking place then

response will take place and if you want to stop that action then antigen

has to be cleared or that cell has to be going into apoptosis and so on.

After binding and cross linking you need a signal to be developed ,and this

signal will be initiated by the clustering process ,and seconds after that,

signal has to pass through ,and we call that (transduction) ,and the signal

passes through the activation of certain proteins that will be present on

these molecules (ITAMS).

so these proteins will get phosphorylated by other proteins and become

activated to attract other proteins like a cascade like the complement and

the blood clotting.

Remember that a cascade is a pathway that is every single step leads to the

next step till we reach the final result.

Finally the activated proteins that will develop will start biochemical

pathways within the cell to amplify the signal so the outcome will be a

cellular response ,and the biochemical reactions will end up in the

activation oftranscription factors.

So transcription and translation protein are going to develop ,and these

proteins will activate the cells, so they will multiply ,differentiate and

produce cytokines.

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(Remember that the most important cytokine is IL-2 which is

differentiation factor produced by T-helper cells, and any suppression of

this factor will have immune suppressive outcome.

If and only if you have missing enzymes in the biochemical pathways thenthe patient is going to have immune suppression_ theSCID syndrome

where you have deficiency in enzymes that could be important in gene

rearrangement like rag1 and rag2 .

Also if there is deficiency in one of the enzymes required for activation

process the same thing will happen).

Sooooo the process starts here by an antigen recognition starting the

response ,then the signal has to be transduced through the activated

(phosphorylated) proteins, and then we will have activation ofbiochemical

pathways in a cascade pattern ,and the outcome of the biochemical

pathways is the activation of gene transcription so differentiation

,proliferation and production of

cytokines will take place.

B-cell Activation:

Lets now look in more details at B

cell (figure 11.1) :

* The antigen has to bind to a

monomer ofIgM ,and of course

there must be a cross-linking ofIgMs

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,one will not be enough.(BCRs are distributed all over the cell surface)

* we have the invariant chains , we have 2: Ig (alfa),Ig (beta).They are

accessory molecules to the BCR that are required for the signal to pass

through , so if they are not present the signal cannot pass through. Theseinvariant chains have specialized proteins (ITAMS) by which the signal is

transduced. By the way (ITAMS) are present in many other cells like T cells.

* the final outcome of the activation and the biochemical pathways in B-

cells and T-cells are the same, the difference is just in the nature of the

proteins that act as messenger molecules for signal transduction events.

T-cell Activation:

If you look at the T-cells (figure 11.2):

* We have the TCR that we have talked

about.

*we have CD3 which is required for the

passage of the signal. Here we have 4

peptide chains on the surface

(gama,delta,epsilon,epsilon) .

* On the other side we have another 2

chains ( zeta ,zeta) also required for the

signal to pass through by the means of

ITAMS , and here _in zeta chains _we

have multiple ITAMS which act as

proteins where the tyrosine is

phosphorylated.

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* phosphorylation of these ITAMS is seem to be very important ,,,but how

they are phosphorylated????

The initiation of the B and the T cell activation starts by thecross linking of

the receptor molecules by multimeric antigen ,and as we said that just one

binding is not enough ,the antigen must react with more than one receptor

at the same time.

B - Cell receptor:

We have membrane immunoglobulin which is a monomer of IgM act as a

receptor. Also we require an invariant protein chain Igand Ig and why we

require those??

>for mIg expression and signal transduction.

>for the ITAM immunoreceptor tyrosine based activation motifs the

protein that are needed to signal to go through .

T - cell receptor:

Its composed of two polypeptide chain or .CD3 invariant protein chains ,

four polypeptide chains two , , and two on the other side we called

them . All have the ITAMs.

the accessory molecule around the receptor are needed because of the ITAMs

that get phosphorylated .

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initiation of B-cell activation:

cross linkinng of receptor molecules by multimeric antigens in B-cells . we have

many B-cell receptor distributed all over the B-cell , and when antigen binding it

will be cross-linked between many receptor.

the second signal is needed we call it co-stimulatory signal .

intitiation of T-cell activation:

T-cell receptor occupation by an antigen on APC presented by class II MHC .Second co-stimulatory signal provided by by APC which could be a cytokine .

Accessory molecule : CD 11a , lymphocyte function antigen (LFA-1) binds to

CD54 or ICAM-1 as ligand and integrin .

So B-cell crosslinking and T-cell binding ag with MHC .

Co-receptor molecules in B-cell activation:

the antigen has to bind more than one receptor (cross-linked ).

Start with invariant chain protein Ig and Ig.

We have accessory molecules on the side :

CD81. CD19 : which only present in B-cell so if I want to target B-cell I can target

the CD19.

CD3 in B and T cellsbind to antigen cross linking in B cell.

CD21 we call it complement receptor 2 , which the same receptor forEBV viruses H type viruseswhich cause infectious mononucleosis . but

here CD21 can bind to CD3 when complement activated .

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All of them they have to be phosphrylated on cytoplasmic tails where the

ITAMs located .

Co- receptor molecules in T cell

activation

CD4 (co-receptor chain) orCD8(co-receptor disulfide

linked dimer) .

CD4 and CD8 areimmunoglobulin supergene

family and they have tails .

CD4 binds to class II MHC. CD8 binds to class I MHC . A protein ofSRCfamily we call it

LCK has to bind to the CD4

,when this is taking place the

CD4 can come closer to class II

MHC.

The same thing here when theLCK binds to CD8 it will come closer toclass I MHC for the activation to take place .

The LCK is associated with TCR if and only ifCD4 or CD8 bind to MHC .Soif genetically you are missing SRC or LCK protein then the process will

stop and you will be immune suppressed .

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Signaling events:PKTs protein tyrosine kinases

and PTPs protein tyrosine

phosphatases as well LYN (in B-cell) and LCK (in T-cell)

phosphorylate the ITAMs .

In B-cell >>When theITAMs in Ig and Ig get

phosphorylated the become

active and bind another protein

called SYk .

Then the SYK will getactivated by LYN .

The SYK will initiate thebiochemichal pathway .

In T-cell >>when the ITAMs inCD3 and chain phosphorylated by

LCK which carried in the tail of CD4 ,

they become active and bind protein

called ZAP 70 .

Then ZAP 70 will getphosphorylated by LCK .

The ZAP 70 will initiate thebiochemichal pathway.

So its all connected as a cascade type

of mechanism of action .

So now we have two protein as an ultimate outcome are going to form the

SYK for B-cell and the ZAP 70 for T-cell .

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Amplification through signaling pathways:

Same pathway inB and T cells

Phosphorylationand activation of

phospholipase C

(PLC), this is triggered

by syk or ZAP70, and

the activation of

phospholipase c will

start the biochemichal

pathways 3 pathways

formation of what we call diacylglycerol and protein kinase C andphosphorylation of several other protein and activating of transcription

factor include NK-B.

formation of inositol 1,4,5 - triphosphate IP3 which lead to release ofstored Ca and level of Ca will increase and this is called influx of Ca in to the

cell and this pathway will activate a protein called calcineurin and the

calcineurin will go and activate transcription factors with in the T helper

cell.

In this step we can use immunosuppressive drugs , like cyclosporin orthe tacrolimus ( these two are the most common used in transplantation

immunology ), they interfere with the production of calcineurin, when you

give these drugs they form the protein or the immunophilins >>> those

interfere with the function of calcineurin >>> so calcineurin cannot be

formed.

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The activation of ZAP 70 or SYK will activate the third pathway whichcalled GTP binding protein or RAS ,Ras activates MAP kinases that

activates transcription factors like AP-1 .

If as I said, any of those enzymes are missing or any of those proteins that

we have like Lyn, Lck, then the patient will be immune-suppressed; and

for example if a patient dont have a protein like in a gamma globulinemia

so patients B-cells will not have the ability to produce immunoglobulin, so

this patient will have immune deficiency, so we will not have humeral

immune response and the only way to help this patient is to give him

immunoglobulins or to treat him with the gene therapy and replace the

infected gene for that particular protein.

Response:

>This is what I was talking about the production of the nuclear factor (NF-

AT, NF-kB, and AP-1) acts on many lymphocytes genes to activate their

transcription.

>This prepare B-cells for proliferation and differentiation and T-cells get

prepared for enhance expression of cytokines as IL-2 IL-2 the most

important.

>Immunosuppressive drugs can modulate this response like cyclosporine &

tacrolimus. You should know these two drugs and there function on

Calcineurin

So this is simple the activation of T or B lymphocytes.

Now we gonna continue the process we have talk about almost all the

adaptive Immune response T-cells, B-cells, antigen recognition molecules

and interactions

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Now we will start talking about the factory; how those lymphoid cells in

general in the second line of defense i.e. the phagocytic cells, the innate,

the adaptive how they develop and we have a comprehensive look into

how those cells co-operate together as one team; when the innate immune

system finish the job it could stop here but if it couldnt then how the

adaptive immune response is going to take place.

Hematopoiesis: CH#12

So these cells we are going to talk about mainly they develop in the bone

marrow and the originating cells we call them the stem cells, and those

stem cells then can differentiate into the main types of cells that we keep

talking about; either the lymphoid cells B, T cells , or the myeloid themacrophages, neutrophils, granulocytes, eosinophils, basophiles, platelets

all of those come from the bone marrow. And these cells develop in the

yolk sack and then they go to the bone marrow after that. So the origin of

the stem cells is the bone marrow

We can get stem cells from the fetal blood i.e. cord blood. And these stem

cells they can reshape and they can provide us with all the different cells

that we need.

How to get these stem cells and how to separate them from others?

How can we separate stem cells, take them, identify, use them to give us

the things that we need?

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By flow cytometry; by the FACS machine you remember florescent

activating cell sorter, those stem cells have special markers that they do not

share with others. One of them is called the CD34; so you can target the

CD34 then u can separate them and use them in therapy.

So the stem cells in order to differentiate and go in many stages, they

require growth factors and cytokines as well, and these growth factors,

colony stimulating factor CSF and cytokines the come from the Stroma

interconnecting tissue of the bone marrow.

And these CSFs you can use them in the lab as well; you can harvest them

and use them as therapy E.g. if a patient bone marrow is suppressed and

cant produce stem cells then I can use CSF to activate the bone marrow to

produce those cells and develop into progenitor cells and then I keep doing

those.

So in each stage we have those growth factors and cytokines that will be

used and finally both cells will develop please look back to the last figure

which is monocytes macrophages, mature cells, lymphocytes,

granulocytes, erythrocytes and platelets and each line of these will require

some of the growth factors and activating factors and so on.

So the process of Hematopoiesis, what do we mean be that?

The process whereby all blood cells are formed i.e. white blood cells,red blood cells.

How those will develop?

Remember the origin is the human bone marrow which is the sourcemeans we activate the bone marrow and all of these cells will come out of

the bone marrow.

The bone marrow is the main factory _remember that_, any suppression

for the bone marrow by tumors for example: leukemia, lymphoma or

use of drugs like chloramphenicol can suppress the bone marrow then

you will be severely immune compromised and you are going to have

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pancytopenia; all the blood elements the platelets, red blood cells,

white blood cells they are going to be suppressed.

Leukocytes Lymphoid + Myeloid.Then Mr. Ali interrupted the lecture and the Doctor said changing places

makes him feel that there is something illegal going on.

And then he added that the most important thing for medical student is to

be honest until improve otherwise role of thumbLOL.

Any try to occupy the seat of others is a crime based on students laws and

regulations. BTW I was one of the criminal in this lecture in order to do this

tafree3 :P .

Back to the lecture:

So we have lymphoid line that will lead to T cells, B cells and another sub of

that is the natural killer cells line. And the myeloid line that gives u

macrophages and the granulocytes and so on.

Then well be talking about lymphoid tissues and organs and cells of the

immune system and you will see how we can classify those into:

>primary organs which is the main factory where the cells will originate

they will be trained and they will get the specific function of a B cell or T

cell; and that is why we call it B cells from the Bone marrow or the bursa of

FabriciusInbirds, the bursa of Fabricius is the site ofhematopoiesis, a specialized

organthat is necessary forB cell development in birds.Mammalsgenerally do not

have an equivalent organ; thebone marrowis often both the site of hematopoiesis

and B cell development.wiki and T cells for the thymus gland. When the cells

go from the bone marrow to the thymus gland they will be trained to

recognize self from non-self and then they will go to:

>secondary lymphoid organs as what we call virgin T lymphocytes ready

for exposure to foreign antigen and then we have the immune response

where the macrophages taking place.

http://en.wikipedia.org/wiki/Birdhttp://en.wikipedia.org/wiki/Birdhttp://en.wikipedia.org/wiki/Birdhttp://en.wikipedia.org/wiki/Hematopoiesishttp://en.wikipedia.org/wiki/Hematopoiesishttp://en.wikipedia.org/wiki/Hematopoiesishttp://en.wikipedia.org/wiki/Organ_%28anatomy%29http://en.wikipedia.org/wiki/Organ_%28anatomy%29http://en.wikipedia.org/wiki/B_cellhttp://en.wikipedia.org/wiki/B_cellhttp://en.wikipedia.org/wiki/B_cellhttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/Bone_marrowhttp://en.wikipedia.org/wiki/Bone_marrowhttp://en.wikipedia.org/wiki/Bone_marrowhttp://en.wikipedia.org/wiki/Bone_marrowhttp://en.wikipedia.org/wiki/Mammalhttp://en.wikipedia.org/wiki/B_cellhttp://en.wikipedia.org/wiki/Organ_%28anatomy%29http://en.wikipedia.org/wiki/Hematopoiesishttp://en.wikipedia.org/wiki/Bird

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Then one of the guys changed his place because someone was sitting on his

seat and the Doctor said it is complicated issue So we will see that how the mother cell originates in the bone marrow B

or T cells; the mother cell or the originator cell of the B cell originate in thebone marrow, lives in the bone marrow, trained in the bone marrow,

differentiate self from non-self also in the bone marrow and then it comes

out to the circulation going to the secondary lymphoid organs, and the

same thing for the T lymphocytes and those which go to the secondary

lymphoid organs where they will be exposed to the antigen.

And then you will see that the B and the T cells they keep circulating

among the secondary lymphoid organs.

And the secondary lymphoid organs are the spleen major secondary

lymphoid organ, lymph nodes all over our body, the adenoids and the

mucosa associated lymphoid tissue MALT.

These lymphoid cells have a target; there main target is to get exposed to

the antigen, if they managed to achieve that role then they will survive,

memory cells will develop and they will stay in our body for a long period of

time.

But if they dont do this function then they will commit suicidal. So all of the

lymphoid cells that come out of the bone marrow and the thymus gland

they will be circulating and trafficking among the secondary lymphoid

organs hopefully that they meat there counter antigen, if they do not then

they will disappear and they will die, and we will tell you more about

Lymphocytes recirculation or trafficking or what we call homing.

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In order to achieve that we will have Adhesion molecules that are present

on the cells that will go through the secondary lymphoid organ, and you

can monitor that through micro-cameras and you can see how these cells

adhere in the mucosal lining and then they can bass through the

endothelial cells into the assigned area of the secondary lymphoid organs

So her in the bone marrow look to the figure we have stem cells and

those stem cells they will keep multiplying and they require activation

molecules, growth factors. And those will go under the influence of the

colony stimulating factors and cytokines in what we call lymphoid and

myeloid progenitor.

progenitor means an intermediate stage cell from HayatStem cells progenitor cells differentiated cells

During that development certain markers are going to be added and some

will be blocks, and we will talk about these, and then they will be

differentiated those from the mother cells into the common progenitor into

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the B cell, the T cell and the myeloid progenitor that gives you the rest of

them development of the erythrocyte and the megakaryocyte and the

granulocytes

The Doctor stopped here and he will continue the next lecture.

Finally

THE END

I wanna Give very big thanks for:

EHAB DEEP: thanks a million brother, actually u did most of the work I

wont forget that

RAF@ TWALBEH: really no words can give you your position in my heart.

MOHAMMAD MEQDADI: I dont wanna thank you because you dont need

thanks :P

And also a very warm thanks for all my friends and to all 2009 batch.

Good luck all In your exams and in your entire life

DrSkodeh