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B-Cell Development Immunology Lecture #12 Colleagues :) just to let you know; the first 4 pages are only

a revision for the last lecture and an introduction for the next two lectures, so either you skip them or read them it's up to you! Last time we were discussing the cells and organs of the immune system. We talked about the primary and secondary lymphoid organs, and the function of each one of them. And finally we talked about the trafficking or homing of the lymphocytes, how those when they come out of the primary, then theyll develop, they will recognize the self from the non-self by the positive and negative selection process. And then theyll come to the secondary lymphoid organs where they are supposed to meet their antigenic counterparts. They will react, then theyll grow and differentiate and produce their effector products whether immunoglobulins or activated lymphocytes; T helper and T cytotoxic. And the activation process as youll see is one arm. When the antigen has to be presented with the MHC through an antigen presenting cell to the T helper cell; this is a one arm or one signal. And well tell you the second signal and from where it is going to come from and the accessory molecules that are going to be involved in this process. And we talked about how those will keep circulating in the secondary lymphoid organs, the lymph vessels, and then the thoracic duct, and then the left subclavian vein in the circulation, and back to the tissues. And they do that once or twice a day hoping to meet their antigenic counterpart, and if they do not, then theyll disintegrate and die! The process takes couple of years in order to commit suicide and die! And we said that what is going to happen if a primary lymphoid organ is shut down, or if it is not there they patient in not born with it-, then the patient will be severely immunocompromized. But if you remove the thymus gland in particular after birth, then here usually nothing is going to happen. While you cannot do that for the bone marrow; because it is involved in other products, then you are going to have consequences related to the other cell products; the red cell line series, neutrophiles and macrophages and platelets as well and other growth factors that are needed.

And we said that those cells as they go through the secondary lymphoid organs they have to be arrested first, and their speed should be reduced. And they do that through the receptors and through the ligands and what we call the selectins. And then after that we have what we call addressins that theyll slow down, and the integrins that come out of the inflammatory process that precedes that process. So new antigens will develop on the endothelial lines like the VLA-4, for example, where it is going to bind to its counterpart on the cell, leukocyte function antigen, ICAM antigens, for example. And then the extravasation is going to take place or the diapedesis, and then they will be near towards the area of infection by what we call the chemotactic factors.
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And those of course in the secondary lymphoid organs that theyll enlarge, and there will be pain and tender related to the interaction that is going to develop; antibodies are going to come out or activated T lymphocytes and memory as well will develop for second encounters. TODAY and tomorrow well be going to talk about how B cells are going to develop. We talked about the activation of B cells and the activation of T cells and how the signal can pass through. But today well talk about how they are going to develop from stem cells till they become mature in the primary lymphoid organs, and when they go to the secondary lymphoid organs, and what are the consequences. Remember that we said when a cell is going to develop; there are certain markers that were going to see. Like in the stem cells the CD34, and when the cells start to grow and develop, those markers are going to disappear and other markers are going to appear that are needed here for the development process, the enzymes, and the gene rearrangements that we have talked about before, or the development of the B cell receptor and the T cell receptor, and how that is going to be modified through the process of self recognition; recognizing the self antigens. So those cells are going to be deleted, and this is what we call the negative selection. And we end up with cells that are only going to recognize foreign antigens and we call that positive selection. Well tell you how the processes of negative and positive selection are going to take place. The negative selection process leads to a term that we always use and we call it tolerance. Tolerance means that an antigen whether it is self or non-self if you dont have a reaction against, then this process is called tolerance. And here we call that antigen tolerogen. And well see how this process of tolerance is going to take place in the primary lymphoid organs in the bone marrow, or in the thymus gland, and we call this central tolerance. Or if any of those cells managed to escape or not to recognize or exposed to self antigens in the thymus gland or in the bone marrow, and how they are going to develop what we call peripheral tolerance.

So I want you to know these terms central tolerance and how we recognize self antigens in the primary lymphoid organs, and peripheral tolerance where these lymphocytes if they manage to escape the primary lymphoid organs. And we still have some antigens that they could be exposed to, and what are the mechanisms that theyll develop, and we call that peripheral tolerance. And you can assure that those lymphocytes are not going to react against self antigens and develop an autoimmune disease. This is the summary of what were going to talk about in those two days. And then well talk about the second signal, the interactions between the lymphocytes; the T and the B through the antigen presenting cells, and the other molecules that are going to be involved and the cytokines. And this will sum up almost ALL the interactions that are between an antigen, antigen presenting cells, and the lymphocytes; and whether they are T cells or B cells.

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And youll see that the relationship between the receptors, the antigenic determinant, the role of MHC, and the cytokines that are involved, and of course the accessory molecules that well be talking about as time will come. So you can see here the process of B cell development from a stem cell into a mature cell, and then plasma cells and memory cell passes through many stages. And during these stages youll see that some markers will appear and some markers will disappear, and the processes that we should know in each step what is going to happen, and if there is a problem in every step, then well going to have a severe immunocompromised condition. So the process starts with the hematopoietic stem cells and the CD34, and then the lymphoid progenerator that will give you the B lymphocytes and of course will give you the natural killer cells as they could be a consequence of that. And then well going to have a pro-B cell that starts to develop here new antigens like CD19, and the CD19 will continue on the surface till the end mature B cell. And then we have the early pre-B cell where the gene rearrangement will start to take place, and the development of the pre-B cell receptor. And well see the involvement of what we call the surrogate chains when they develop into the late pre-B cell. Early and pre where the gene rearrangement will put on hold, because they just want one new gene to be rearranged, and well have just one specificity. And if they keep doing that, well have more than one specificity on the surface, but that doesnt happen this way! So youll see how the gene rearrangement will be upregulated and downregulated during that process; early to late. So the outcome here is the development of what we call the immature B cell, this is ALL in the bone marrow. And youre going to see how this B cell receptor is going to develop, and the process by which they are going to recognize the self antigens, so theyll be deleted or theyll be in a frozen state, and we call that anergy.

And then theyll come out as mature cells into the blood and then into the secondary lymphoid organs; where they are going to develop into plasma cells or into a memory cells, and then the memory cells will go into the plasma cell immediately, and theyll give you the antibody that is involved. And then well see when the immature B cell that is still in the bone marrow recognizing a self antigen, then theyll try to change, and we call that receptor editing. So they try to change and to switch that specificity, so if theyre successful, then it will continue and go to the secondary lymphoid organs, and if it fails, then that is supposed to be deleted, or they could go into the circulation where other mechanisms in the peripheral tolerance where they could delete those as well see. So well notice that the change for the tolerance, the central tolerance that occurs at the receptor is going to involve the light chain. So the light chain whether it is Kappa or lambda; it plays a role in trying to switch or re-edit or to change its specificity and not to bind to self antigens as were going to see.
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So let's start now talking in more details :)

B-Cell Development The process of the B cell development starts from the area close to the bone; the peripheral part (the endosteal surface) and goes toward the central part (central marrow space) where we will have the mature B cell. Figure 14.1: very close to the bone you can see hematopoietic stem cells marked by CD34, and then they will start to develop into the progenitors; pro-B cells then the pre-B cells where the BCR starts to develop. And there is a light chainlike; called surrogate light chain (its not the original one, you will read about it in a seconds), and then you have macrophages; they are needed for the negative selection process to take place, they will phagocytose all those that are supposed to react against self antigens. And they will end up having immature B cells, then they will go to the circulation as mature cells to the secondary lymphoid organs. So the process goes this way from the periphery to the central, around almost 80% of these lymphocytes that start developing will be deleted, because they will recognize self antigens, so less than 20% will go to the secondary lymphoid organs (In T lymphocytes more than 95% will be deleted so we end up with less than 5% that will be selected to go to the secondary lymphoid organs).

Figure14.2: look at the surface markers; we start with the CD34 present only on the hematopoietic stem cell and maybe on the pro-B cell, after development it will disappear. CD19 doesn't present on the stem cell but starts to develop at the pro-B cell and continues all the way till the mature B cell. CD19 can be excellent marker to separate Bcells from T-cells. Expression of RAG genes; at the stem cells nothing is going to happen, process starts at the pro-B cell, it will start with JH and DH, so the heavy chain starts to rearrange and then surrogate light chain (L ) will be added and then the light chain at the end of the immature level. So their expression will start to form the heavy chain then they will be downregulated during early pre-B cell stage when VH and DHJH are rearranged to ensure that DHJH wont select another specificity, then expression of RAG genes will be upregulated again to form the light chain at the late pre-B cell stage and at the immature they will stop. Adding of the constant region will happen at the late pre-B cell; C will be selected thus IgM will be the first Ig to be produced. The CL will be added finally at the immature state. Earlier there we will have surrogate light chain (L) that will help this process to shape the BCR, this L will start to develop at the pro-B cell and continues to the early pre-B cell, so this surrogate light chain will develop temporarily to help the heavy chain and it will disappear at the late pre-B cell when the rearrangement of the light chain is going to develop (VL to JL rearrangement). Notice when we have the surrogate light chain the receptor will be called

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pre-BCR, and we will not have complete BCR until the cell is going to the secondary lymphoid organs as immature cell. The significance of the BCR at the immature that it still has the chance to change its specificity through the receptor editing process if they recognize self antigen before going to the lymphoid organs where they will not be able to edit anymore. B-Cell development mIg (membrane Ig) and BCR genes rearrangement at the germline level. Then the pro-B cell. Negative selection process occurs during which only positively-selected cells will end up going to the circulation, if this process failed autoimmunity will develop. Positivelyselected cells will go to the secondary lymphoid organs where they can meet their antigenic counterparts, terminally differentiated to antibody producing cells. If the cell didnt encounter antigen they are going to die after maybe 1 year, so it will take a chance to be exposed to foreign antigen. Then pre-B cell early and late. Immature B cells. Then cell goes to the circulation as mature B cells and finally they will change to plasma or memory cells. Stem cell to Immature cell The scenario starts from yolk sac to the fetal liver then to the bone marrow; immature to peripheral and more mature cells to the central part, stromal cells contribute growth factors that are needed for the process of development e.g. IL-7 that plays a major role in gene rearrangement. Pro-B is the earliest stage; starts with the CD19, DHJH to VH rearrangement then expression will follow. Then the early pre-B where the surrogate light chain will be added surrounding the C , Ig & Ig for the signal to pass through pre-BCR. At this stage there is suppression of RAG1 & RAG2 and TdT to ensure we will obtain only one specificity, if they remain active we could develop more than one specificity and if those enzymes arent present we could end up with severe combined immune deficiency. Late pre-B where RAG genes are upregulated again to produce VL to JL rearrangement. The immature B cell which is the final stage where the actual light chain & will be added, so this is the immature BCR that could go into receptor editing as was mentioned before, positively-selected cells in the negative selection process will go into mature cells to the peripheral lymphoid organs. In this process one specificity will come out, it comes either from the father or from the mother in a process we call allelic exclusion; only one allelic form will come out; (not like the MHC) both allele 1 and allele 2 will be rearranged but only one will be shifted, the process of choosing one allele and not the other one is not really clear.

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Figure 14.3: if the first way failed, for example, then the other one will continue, if neither of them succeeded the process will stop and the cell is going to die. Figure 14.4: in the light chain we could have & ; if V1kJ1k proceeded then we will get light chain with certain specificity, if it didnt continue then V2kJ2k may continue and we will get another light chain with another specificity, if not they will try the , so we will end up with one allele either from the father or from the mother with particular specificity, if neither of the alleles succeeded to continue, then the cell will die. * Allelic exclusion: one specificity from the mother or from the father is going to be expressed, this is related to both heavy and light chains * Ordered Ig gene segment rearrangement and allelic exclusion - One heavy chain with one type of light chain, one parental chromosome is going to be expressed at a time (allelic exclusion), this result in just having one specificity on a cell (clonal selection theory), otherwise more than one specificity will arise on the same cell and we don't want that to happen of course! What about the IgM and IgD? When the immature B cell develops, it starts with IgM antibody on the surface, and when it goes to the secondary lymphoid organs, the IgD is going to be expressed more. So the more IgD you are going to have on the surface of the cell, the more maturity of B cell, the more age, the more development, and vice versa. So the first Ig that will appear on the surface of the immature B cells is the IgM, transitional stage B cell: surface IgM and then it starts to develop the IgD and when they go ahead to the mature form we will have both. And remember: IgM and IgD are co-expressed on the surface by alternative splicing starts with the IgM first and then the IgD, so they are both going to be expressed on the surface.

Immatue to Mature B-cell transition This is a very important step in the positive and negative selection where the cell still have a chance to re-edit the receptor; the IgM and IgD. And the editing process will only involve the light chain.

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During the development from immature to mature the enzyme RAG-1 and RAG-2 will be downregulated; it makes sense when we want to have one specificity, the surface IgM expression increased, surface IgD begins to appear, and finally it ends up with mature B cell having IgM and IgD on the surface. This mature one which will go to the secondary lymphoid organs we call it now Nave or virgin B cell where it will meet its antigenic counterpart. And when it meets its antigenic counterpart the first time we call that cell primed. We can study the gene rearrangements through PCR and we can use it to make diagnosis of malignancies related to B cells. For example, this is a control monoclonal B cells, this is lymphnode DNA control showing polyclonal ladder, this is patient sample 1 with Bcell lymphoma, this is patient sample 2 with another B-cell lymphoma, and in lane 4 you can see the other genes but the one dominant monoclonal product can be seen here, and we can know what is that particular gene that is responsible for that malignancy since we have the primer and that primer can be labeled. Nowadays, we don't do that anymore, we rely on the cell markers that appear on the surface of those cells.

This is the process of negative selection. Look at the development of the immature B cell to mature B cell that has the surface IgM and IgD. In the immature form the IgD has not developed yet. But how those cells are going to be negatively-selected? We can see here two
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processes that are going to take place: the first one- if the receptor is going to be crosslinked with self antigens, it is going to make a deletion for the B cell. The second one- if the antigen is soluble and the B-cell receptor can be exposed to that then the cell will go into a state called anergy; the cell is going to be frozen. So here we have multivalent cell surface self antigen where extensive BCR cross-linking takes place and when that happens then maturational arrest is going to take place and receptor editing tries to fix the problem. If it was successful then it will be positively-selected and if it fails the cell is going to die. And the same applies if we have high dose of soluble self antigens where we have limited B-cell cross-linking and then functional inactivation of anergy is going to take place. The difference between the immature and the mature is only the receptor editing; where it can take place in the immature while it cannot in the mature. This is really interesting here where the negative selection process is going to take place in the bone marrow when those immature B cells are exposed to self antigens, during that process, if you inject an antigen to the bone marrow and those immature B cells reacted against that one they will consider that as a self antigen and the receptor will be edited and that antigen is going to be selected as self antigen although it is a foreign antigen. So any antigen the B cell get exposed to during its development, it will be considered as self antigen and it will be recognized in the future as self antigen and that strains against that particular antigen are going to be deleted. And you can exchange grafts of that particular antigen that you have inserted in the early development. That is really interesting! ^^ Figure 14.8: This is how the process of the receptor editing is going to take place and remember that this process is going to take place through the light chain only; either kappa or lambda. So here for example, this is the immature B cell and we have cross-linking of self antigen so what's going to happen? You see here the kappa light chain genes; V and J where gene rearrangement is going to take place and one is going to be selected. If we already have V1, for example, we will try to have another one, say V2, in attempt to change that particular self-reactive specificity and to change that receptor from a one that can bind self antigens to one that cannot. If it was successful then that cell is going to be positivelyselected, and if it wasn't successful, it will try again, but if it again fails then that cell is going to be deleted. Receptor editing mechanism is one of the mechanisms that B cells use to be so evasive from binding to self antigens.

Induction of Tolerance Negative selection: Deleted or functionally inactive B cells when encounter self antigen.

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Tolerance: it is when B cells do not respond when challenged with self antigens; it is the process of not-recognizing self antigen. We said that we have central tolerance where the recognition is going to take place in the primary lymphoid organs OR peripheral tolerance when the recognition is going to take place in the secondary lymphoid organs. Tolerance is needed to prevent autoimmune diseases. Some microorganisms manage to disguise themselves as self antigens, so they will be tolerant to our immune cells, like schistosomes which cover themselves with self antigens e.g. blood antigens, MHC antigens, so they will be recognized as self. BCR cross-linking and antigen dose are which will determine the mechanism of tolerance; cross-linking the receptors together or the antigen dose is high. Again and again :D Receptor editing following V(D)J rearrangements is by changing the light chain. Clonal deletion: if that clone recognizes self antigen then it is going to be deleted. Polyvalent VS monovalent antigen and dose: Polyvalent that will cross-link, and monovalent where the dose is high and it will lead to anergy. Only 20% of B cells survive to periphery and 80% is going to be negatively-selected.

Additional Tolerance Induction (peripheral tolerance- The mature B cell line) If it wasn't completely successful at the central level since not all self antigens are present in Bone marrow; then we could have some B cells that can react against self antigens in the circulation. Antigen engagement is crucial for survival, otherwise deletion or anergy develops. Multivalent antigen induces cell deletion while monovalent induces anergy. When sIgM and sIgD develops no change occurs. When an antigen is presented with an antigen presenting cell then the process is going to be very effective and the memory cell will develop and that particular antigen will go through this process of antigen presentation and so on, we call that thymus-dependent. We mean by thymus dependent; the involvement of T-helper cells. And this is crucial for isotype switching and the development of memory cells. Isotype switching is the development of immunoglobulins other than IgM. This gives more efficiency for the system.

Figure 14.9: Look here for the mature IgM and the IgD antibody on the B-cell: when the antibody is bound to IgM and IgD its going to be taken inside the cell, processed and
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presented to the T-helper cell. So the T-helper cell is going to be activated to produce the cytokines, then the cell can develop into a plasma cell and antibodies will develop and then they can go into Improved B-cell (B-cell with improved BCR) and memory cells will develop. When the memory cells are already have exposed to antigen, the time is going to be shorter and more efficient. So this is the Thymus Depenedent Anigens. This process occurs in the secondary lymphoid organs; lymph nodes or spleen, and thats why these organs get enlarged. Activation and antibody production It occurs in the secondary lymphoid organs where there are antigen-presenting cells. Remember that we have T-cell dependent area where there are T helper-cells. B-cells keep circulating till they meet their antigenic counterparts and become prime, if they don't, the B-cell is going to disintegrate and die after a period of time. Remember that! The role of T helper is extremely important because it provides the process with cytokines, lymphokines and MHC antigen class 2 which is required for antigen presentation on the antigen presenting cells. Thymus dependent antigens vs. thymus independent antigens: when a B-cell encounters an antigen and can be triggered, like when we have certain polysaccharide antigens that they cross-link the surface of the B-cell and trigger it, itll only produce IgM antibodies, no memory cells and no isotype switching, while if the process involves antigen presenting, class 2 MHC antigens, T helper and the cytokines then they are going to develop into memory cells and we'll have isotype switching (IgG antibodies and other antibodies). So the process is extremely efficient with the thymus dependent. Continued Selection of B cells in Lymphoid follicles Continued selection of B-cells in the lymphoid follicles is required for signal induction; the receptor has to be occupied. Primary follicles; the first time they occur in the lymph nodes. Secondary follicle is the second time. And the fate of antigen encountering is plasma cells and memory cells development. Germinal centers, affinity maturation and class switching: remember the somatic hypermutation for a better fit, so the quality of IGs that will develop in the thymus dependent are going to be much better. Which is called it affinity maturation.

Plasma cells dont respond to antigens but memory cells do so, they can develop into plasma cells. Plasma cells dont have receptors on their surfaces, their function is just to produce the Igs. Figure 14.10: This is what happens in the secondary lymphoid organs as you can see in the assigned area. Here you have whats called follicular dendritic cells that present the antigen
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at their surfaces, but these cells dont have class 2; they have an Fc receptor and complement receptor that binds the antibody and the antigen if they are immune complexes. And then you have the B-cell and its receptor that are going to bind, theyll be activated and this is another mechanism of activation then plasma cells and memory cells development. So an APC is crucial for that particular process, and it occurs in the B-cell zone.

Thymus-Independent Antigens Activation without B-cell help, no cytokines involvement and no class 2 MHC. The second signal is derived from the antigen itself. Examples on thymus-independent antigens are repeating polymers antigens, bacterial polysaccharides, dextran and lipopolysaccharides. Fast response when needed. But the thymus dependent is more effective process. Figure 14.11: Again, this is the thymus independent. Just cross linking of the antigen receptors (surface IgM) would be enough to induce a signal, this signal will produce IgM antibody only without affinity maturation, without isotype switching. B cell repertoire during human cell development When the B-cells start to develop and the development of IgD antibodies, the B-cells that develop early in life differ from the B-cells that adults have. For example the B-cells that are developed in fetal life are called B1-cells, but the ones that develop later in life are called B2-conventional cells and they have the IgD and they are more mature. When B-cells develop they have a CD5 marker which is presented only on early B-cells, its supposed to disappear when we grow up, if it fails to disappear then this is a marker of malignancy (chronic lymphocytic leukemia). They have noticed that the type of the Ig that developed by the fetal B-cell early in life are called natural antibodies. These natural antibodies have a broad specificity compared to that of conventional B-lymphocytes antibodies that develop later in life, and it makes sense here: you need immunoglobulins with broad specificity early in life compared with immunoglobulins with high specificity in later in life. We have limited specificity early in life, few variable heavy genes, lack of TdT, so they dont change that frequent. The B-cell receptor of B1 type cross reacted with many antigen specificities, thats why they are called natural antibodies.

Introduction to Chapter 15 - T-cell development

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The T-cell development is exactly the same as the B-cell development but the difference is this occurs in the thymus gland and theres no allelic exclusion. Here we have the stem cells that came from bone marrow, it goes to the thymus gland and start to develop in the periphery as thymocytes and then they keep going to the cortex and then into the medulla (Subcapsular, cortical then to the medulla). The same mechanism that happened in the bone marrow is going to happen here again; negative selection process which is in the B-cell development happened by exposure to self antigens and in the T-cell it happens by exposure to class 1 and 2 MHC antigens. There are two points about negative and positive selection: 1) Exposure to self MHC antigen (MHC restriction). 2) Binding to self antigens as well (tolerance). We are talking about MHC class 1 and 2 and to which one the cell is going to bind whether its a T helper or T cytotoxic cell. The MHC class will determine the type of T-cell bound. Now when they go to the medulla; all of the negative selection are going to take place (98% are going to be deleted), only 2% are going to escape from the thymus, they are going to help B-cells and T-cells, they can cause destruction to cells through T cytotoxic cells and they could activate inflammation through production of cytokines and chemotactic factors. How diverse populations of T-cells with different specificities are going to develop? Positive and negative selection, T-cells develop in the thymus gland, thymocytes mature to mature T-cells, 98% of them die because of the negative selection process, remember that! Self tolerant and self restricted; we need them to react against foreign antigens with the self MHC antigens. You have to be familiar with these terms! Self tolerance: not reactive against cell antigens. Self restricted: the T-lymphocyte should be used with the same MHC antigen.

LIFE
Isn't about waiting for the storm to passIt's learning to DANCE

IN THE RAIN!

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