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Lymphocyte activation and hematopoiesis Saad Aldeen Mohammed Ziad Al-Nasser Wednesday, 13/7/2011

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).

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 Tcytotoxic 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

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 of transcription 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.

(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 then the patient is going to have immune suppression_ the SCID 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 of biochemical 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 of IgM ,and of course there must be a cross-linking of IgMs

,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. These invariant 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 Bcells 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.

* 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 the cross 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 Ig and 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 .

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 cross linking 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 cells bind to antigen cross linking in B cell. CD21 we call it complement receptor 2 , which the same receptor for EBV viruses H type viruseswhich cause infectious mononucleosis . but here CD21 can bind to CD3 when complement activated .

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) or CD8(co-receptor disulfide linked dimer) . CD4 and CD8 are immunoglobulin supergene family and they have tails . CD4 binds to class II MHC. CD8 binds to class I MHC . A protein of SRC family 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 the LCK binds to CD8 it will come closer to class I MHC for the activation to take place . The LCK is associated with TCR if and only if CD4 or CD8 bind to MHC .So if genetically you are missing SRC or LCK protein then the process will stop and you will be immune suppressed .

Signaling events:
PKTs protein tyrosine kinases and PTPs protein tyrosine phosphatases as well LYN (in Bcell) and LCK (in T-cell) phosphorylate the ITAMs . In B-cell >>When the ITAMs in Ig and Ig get phosphorylated the become active and bind another protein called SYk . Then the SYK will get activated by LYN . The SYK will initiate the biochemichal pathway .

In T-cell >>when the ITAMs in CD3 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 get phosphorylated by LCK . The ZAP 70 will initiate the biochemichal 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 .

Amplification through signaling pathways:

Same pathway in B and T cells Phosphorylation and 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 and phosphorylation of several other protein and activating of transcription factor include NK-B.

formation of inositol 1,4,5 - triphosphate IP3 which lead to release of stored 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 or the 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.

 The activation of ZAP 70 or SYK will activate the third pathway which called 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 (NFAT, 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

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 the macrophages, 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?

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 source means 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

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 thumb LOL. 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 Fabricius In birds, the bursa of Fabricius is the site of hematopoiesis, a specialized
organ that is necessary for B cell development in birds. Mammals generally do not have an equivalent organ; the bone marrow is 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.

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 the bone 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.

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 Hayat Stem 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

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