Adaptive Immune Cells PDF

parole Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 ADAPTIVE IMMUNE CELLS The innate immune system is activated immediately when a pathogen tries to enter our body. Sometimes it is enough to destroy a pathogen, but if ther...

parole Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 ADAPTIVE IMMUNE CELLS The innate immune system is activated immediately when a pathogen tries to enter our body. Sometimes it is enough to destroy a pathogen, but if there are many pathogens, a strong inflammation occurs and activate the adaptive immune response, which is our second line of defense. This is made of lymphocytes, that are mainly T and B cells and in today lessons we will see which are the main features of these cells, the differences between these cells and the one of the innate immunity. pepraedal patogeno On the surface of our cells we have the MHC complex that present the peptides to the circulating cells, also this complex is very important because it represents the evolution of our system. Some pathogens evolved and became able to go inside our cells, so in this way our circulating cells cannot detect them. To see what happen inside our cells, the immune system developed the MHC complex. We have 2 MHC complexes: class I and class II, for example here we have different MHC. Some cells o can express both MHC I and MHC II, other cells can express just one class. MHC II is able to present also self-peptide, coming from degraded old proteins. In MHC complex we have the presentation of peptide from pathogens. Here we have a pathogen (a virus or a bacteria) that can enter into our cells and some peptide of this pathogens can be presented in MHC complex. For example the peptide from pathogen are the pink one and these peptides compete with the normal endogenous one, because in this way our cells of the adaptive immunity can see them and try to eliminate them. When the infection progresses the peptides from pathogens became more expressed than the normal endogenous one. As we can see, the pink peptides became more numerous than the green one, that are the self-one. 1  Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 self iniziano adiminuire g 2º1 III adf.ie Ipreou Now that we have all these peptides present in the MHC when the T lymphocytes, which are the sensors of the adaptive immunity, arrive and with the TCR (T Cell Receptor) made of 2 chains, 𝛼 and β chain, these T cells start touching the surface of all the cells trying to find something strange like some peptides from pathogens that can activate them, and they try to destroy the infected cells. If the T cells find a pathogen peptide after a short communication between the peptide and the T cell that become strictly connected, in this way the T cells can activate themselves. What are the effects? We have the activation of the cells and we can have the release, for example, of perforin and granzyme that can kill the cells. The CD8 cells are able to kill the cells with the release of granzyme and perforin, differently from the CD4 that release cytokines and has a helper function: they are involved in the cooperation of T cells, trying also to activate a humoral response. The adaptive immunity is the second line of defense because we have first the body barriers. The different mechanisms of the innate immunity can offer protection against the pathogens, but also pathogens evolve because they live very well in our cells (they have an optimal temperature, all the nutrients to survive, protected from the external environments trying to kill them). The system so evolved trying to expose on the membrane of our cells what happen inside the cells: this is the function of the MHC, which is very important to I activate T lymphocytes, because they need the presentation of the peptide by the MHC class complex. Without this complex they are not able to nsmnie CD8 recognize the pathogen. 2 class 1 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 Which are the features of the adaptive immune response? GCR orBCR 1) We have an enormous number of receptor different from one T cell to another one, that is very specific for the peptides that they can bind. Thanks to the high number of receptors, we could be able to recognize all the pathogens, also the one we have never been exposed in our life. The immune system try to give us alle the possibilities to defend against all the pathogens, just to be prepared for any encounter. 2) The clonal expansion of the cells (lymphocytes) that have the specific receptor that recognize that specific peptide presented in MHC I / II in case of T lymphocytes. Just the lymphocytes that recognize the specific peptide can expand. 3) Acquisition of an enduring memory. After the battle some cells that were expanded will survive longer (also years) and they are called memory cells. They are very important because thanks to them we have developed the vaccine (we can use the vaccine strategy thanks to the memory cells). We have these stem cells that can originate different cells with a high number of different receptors (around 1011). There are different lymphocytes with different receptors. Some receptors can bind the self-antigen of course (we don’t understand what she said) pro-apoptosis, otherwise we could have auto-immune diseases. Just the peptide that do not bind with high affinity self-peptides will survive. If an antigen from a pathogen arrive this will be bound to the receptor more similar to it. In this case we have this lymphocytes that can enter perfectly just in that lymphocytes and just these cells expand. All this cells can help in the eradication of the infection. We need this lymphocytes to eliminate this antigen, we need a lot of these cells to destroy the pathogen. 3 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 The cells of the adaptive immunity are T and B cells, that can specifically bind an antigen, we have also the possibility to have cells that can recombine genes coding for the antigen receptor. Thanks to the recombination we can have many different receptors. The image shows the structure of the T lymphocytes, they have a big nucleus and a few cytoplasm around it and on the membrane of the cells there are: 1) TCR receptors, which are very important to interact with the HLA complex with the peptide. 2) CD4 or CD8 co-receptors 3) The CD3 chains, that are very important for the signaling transduction 4) Adhesion molecules which are very important because they allow the crosstalk between the lymphocytes and the antigen presenting cell. 5) Receptors important for the decision fate of the cell, the fate receptors 6) Hormone receptors 7) Cytokine and chemokine receptors because the lymphocytes need to know where the pathogen is or in which part of the lymph nodes it will be and the cytokine receptors because the lymphocyte needs to know if it need to be expanded or not. The B cells have, as the T cell, a big nucleus and the cytoplasm around it, also they have on the surface: 1) BCR can bind the antigen without the presentation in MHC class I or II (the first big difference compared to the T cells). 2) Co-receptors. 3) Adhesion molecules because B cells can present also the peptide. B cells can bind the pathogen, endocyte it and present it to T cells. To do this it need the adhesion molecules to permit the crosstalk between cells. 4) Lymphocytes also need to know where they have to go, if they need to be expanded or not, and for this they have cytokine and chemokine receptor. 4 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 In the evolution of our immune system this is not enough because our immune system needs to know with precision if the lymphocytes need to be expanded or not, so to have the expansion and the functional activation of these cells we need also other signals delivered by other cells. The reaction of activation of T and B cells may inhibit or kill the pathogen directly, but these cells have the ability to recruit many other cells and immune mechanisms. First, we can have a humoral or cellular immunity: when we have an extracellular microbe, this, through the activation of B cells (that became plasma cells to secret antibodies, which can bind this microbe extracellular) we can have the inhibition of the infection and eliminate the microbe. Extracellular microbe can be phagocytes and can survive into the macrophages, in this case we have the activation of T helper cells that can activate macrophages or neutrophils through the secretion of cytokines and in this way the activated phagocytes can kill the microbe. However, the microbe can be also inside our cells, like viruses, which can use our biological machinery to replicate themselves. In this case we have the activation of cytotoxic T cells that can directly kill the infected cells and eliminate the reservoir of the infection, using perforin and granzyme. bianchi nucapi 9 complement Phagocitosi's acquiang About the time of response, different phases are present: recognition, activation, antigen elimination, decline of the response, survive of the memory cells. 5 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 decrease Iin limpronades giorni As you can see in the x axis all this process is quite long because first, we need to have B lymphocytes that need to bind the antigen or T lymphocytes that thanks to the antigen presenting cells recognize the peptides that is presented in MHC complex. After the recognition we have the clonal expansion of T and B cells and then the differentiation of the B cells who differentiate in plasma cells that can produce antibodies, or lymphocytes can differentiate in effector T cells that try to kill the pathogen. Antibodies and effector T cells try to eliminate the antigen and when they finish their function, we have this decline of the response. The cells that collaborate to the antigen elimination will underwent to apoptosis and just few of them will also survive some years and they will become memory cells. The first thing we see in the graph is that to have the antigen elimination, so the active phase which can destroy the pathogen, we need about 10 days, so in some days our immune system needs to start all the process. Which are and from which cells originate B and T cells? From the hematopoietic cells originate the myelinated stem cells from which originate all the immune stem cells and through the influence of bone marrow stroma cells on a lymphoid stem cells. These cells can be differentiated in B progenitor, that in bone marrow will give the B cells, while the T progenitor through the education into the thymus can originate CD4 or CD8 T cells or NK cells. 6 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 già PARLATO ADAPTIE minima How can we understand if B cells became a T or a B lymphocytes? o The most important signal is Notch I, which plays a critical role in this decision. A positive signaling from notch will drive the progenitor into the thymus and we will have CD4 and CD8 T cells, while the absence of the positive signaling of notch will drive the progenitor cells to the bone marrow and so to the B cells or to the formation of NK cells. 7 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 Primary lymphoid organs, like the bone marrow or the thymus, are the organs in which we have the I formation of the B and the T lymphocytes, but then the cells through the blood and through the lymph will reach secondary lymphoid organ, constituted by lymph nodes, spleen, and mucosa lymphoid tissue. The mucosa associated lymphoid system that are mainly present in the lung, gut, and skin because they are the most exposed to the pathogen (for example when we breath or eat we allow the entrance of them), so this is why it is a very important defense against the pathogens. Other secondary lymphoid organs are the tonsils, the adenoids, the appendix and the Peyer’s patches. 1 8  Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 ENTITA Usurari INIBITI Here we have a draw of the lymph nodes. They are made of different, afferent, and efferent lymphatic vessels (the way of entrance and exit), also there are different septum that divide the lymph nodes in different sections. We have the primary follicle in which we can also have germinal center and secondary follicles, in this sides both primary, secondary, and germinal center we will find the B lymphocytes. Just around them, in the cortical area we will have the T lymphocytes. This is an immunohistochemical staining that shows you, in brown, the positive cells for CD3: the T cells. The T cells are just around the follicle. How can the T cells go only in this part of the lymph nodes? Thanks to this chemokine that can drive the T lymphocytes in this area. 9 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 These are the lymph nodes that are stained for CD20 that is a common marker that is used to identify B cells. They are in the follicle also in this case thanks to the chemokine that again can drive B cells. How can B and T cells patrol our body? The microbe can be captured by an antigen presenting cells, that thanks to the inflammatory cytokines, can lose the adhesiveness to the tissue and can start migrating into the lymph nodes where we have the T and B cells. How can B and T cells can reach the lymph nodes? Thanks to chemokine and a particular structure, called high endothelial venule. 10 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 This structures are specialized blood vessels that allow the blood circulating lymphocytes to enter into the lymph nodes. This can be possible thanks to the presence of L-Selectin, LFA-1 and CCR7. Naive lymphocytes express a lot of L-selectin on their membrane because activated lymphocytes do not express L-selectin, which is enzymatically degraded. Only the naive lymphocytes, that are lymphocytes that never encountered the antigen, express a lot of selectins, however when they are activated, they do not express them. This electronic microscopy image shows us the T cells adhesion, also the contact with specialized blood vessel we said before (specialized endothelial anodes). 11 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 DAiLYIFEFmpymI Gemme mator Pekin Fenzi If the naive T cells recognize a peptide presented by MHC II, these T cells become the activated T cells (red one) and they can exit the lymph nodes through the efferent lymphatic vessel and through the thoracic duct and reach the peripheral tissue where the pathogen is present and do their job (if it is CD8 will kill infected cells or if it is CD4 it will secrete cytokines). Which are the regulators of the lymphocytes in the circulation in the lymphoid tissues? There are different molecules: 1) The selectin L control the naïve lymphocytes arrival into the tissue. 2) Different chemokines (and chemokine receptors) that control the movement of these lymphocytes and drive them into specific areas (CCR7-CCL19 and CCL21). 3) Sphingosine I phosphate receptor (S1P) control the exit from the lymphoid tissue. This is very important because it is inhibited after the lymphocyte’s activation thanks to the presence of interferon type I (IFN I) and CD69. How does it happen? The sphingosine I phosphate is a lipid chemoattractant factor that is abundant in the blood and less into the tissue. When a naive T lymphocytes is circulating, it expresses low levels of the receptor because the receptor is internalized inside the cells, while after they enter into the lymph nodes, they re-express the receptor and thanks to the rich expression of the receptor, the lymphocytes can exit from the lymph 12 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 nodes because they bound the sphingosines that is abundant in the blood. If the T cells encounter the specific antigen, T cells need to remain into the lymph nodes and so the activation inhibits the expression of the receptors in the surface, in this way the lymphocytes may rest into the lymph nodes because the lymphocytes recognize something strange and now cannot exit, because they need to be activated. After the full differentiation the receptor is expressed on the cell surface (it will take about 2 days) and activated lymphocytes can exit. The naïve T cells just enter into the lymph nodes through the specialized blood vessel. In green is indicated the sphingosine receptor, which is inside the lymphocytes. If we have an antigen recognition the T cells will be activated and, after the activation, the receptor for the sphingosine will be exposed on the membrane of the T cells. In this way the T cells can bind the sphingosine coming from the blood and slowly can exit as an activated T cell. This also may not happen if the T cells with the inside receptor do not recognize any antigen, the naïve T cells express immediately the receptor on the surface and in this way, it can bind the sphingosine a slowly exit binding the lymph nodes as a naïve T cells. This naive T cell can reach other secondary lymphoid organs trying to find some antigen that can be recognize. Potentially our lymphocytes can check all our secondary lymphoid organs daily trying to find an antigen to recognize. If the T cell is activated it will reach the site of infection. 13 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 If we have B cells more or less we have the same things: the B cells can enter to the lymph nodes thanks to the specialized blood vessel, we have some chemokine and chemokine receptor that can be activated and can drive the B lymphocytes and if they encounter the antigen can become an activated B cells, from which we can have the plasma blast, that can exit from the lymph nodes, and through the blood circulation we can have the production of IgG from cells that migrate to the bone marrow, or the production of IgA. When we talk about the different function of antibody isotype, it will be clearer the different function that we can have if we are in presence of an IgG or an IgA. Basically, the mechanisms are the same. T cells 14 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 Looking more in the detail the T cells, as we already have said, they have a big nucleus, few cytoplasm, some mitochondrial, the ER (endoplasmic reticulum), the lysosomes. They called T cells because they mature in the thymus. We also have a lot of T cell subpopulations: 1) The CD4, helper cells that secrete cytokines which are very important for the cooperation of T and B cells 2) The CD4 regulatory T cells are very important for the selection of the expansion of the clonal cells. They are involved in the elimination of the cells that potentially can recognize self-peptides, from which we can have autoimmunity problems. So the important function is to eliminate those cells that recognize themselves. 3) The CD8, killer cells 4) The CD3 + and CD8 + cells are associated to the mucosae 5) NKT cells, that are positive for CD3 6) T cells CD8 + or CD4 +, but gamma-delta. The difference with other one is that those cells have a TCR made of 2 different chains (not the alpha-beta like the CD4 or the CD8), but is formed by the gamma-delta chain The T cells, like the B cells, derive from a multipotent bone marrow cells that guide the differentiation. From the bone marrow immature T cells are able to migrate into the thymus, where they differentiate and acquire the specific receptor. Only the T cells that express a TCR are able to interact with low affinity with the self HLA antigen and they will exit from the thymus and enter in the blood. Remember: just the one that interact with low affinity, not the one that not interact or the one that interact with high affinity, this is important to avoid the development of autoimmunity disease. The T cells, emerging from the thymus, are naïve (or virgin) T cells that express a high number of TCR (Something about 105) are able to react with poor affinity with the self HLA molecules, in which we can 15 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 have self-peptide. Another important thing is that each virgin T cell express the TCR with different binding site, because otherwise we can recognize just one antigen. Once activated, it will generate a clone of T cells all with the same TCR. So basically the TCR is the majority of the T lymphocytes, it is made of the alpha-beta chain and we have also the CD3 chains that rapporto are important for the transduction of the signals. The other important thing is that the ratio between CD4 and CD8 T cells is about 2:1 (two to one) and this can be very important. For example, if we have a patient that develop a lot of infection and he will require many time to destroy the infection; one clinical examination we can do is to analyze the ratio between the number of lymphocytes (we can use the CD3 for this) just to see if the total number is correct, but then we can check also for the ratio of CD4 and CD8 that sometimes can explain the development of some pathologies. The T cells, that express CD4 co-receptor interact with class II molecules while the T cells, that express CD8 co-receptor, interact with class I molecules. Here you can see this interaction, there is the target cell, the HLA class I in which you can see the alpha chain and the beta II microglobulin (the alpha chain is the most polymorphic one) and between the domain alpha I and alpha II we have the molecular pocket in which we can see the peptide that is presented to the T cells. In this case we have the CD8 cells because we have a class I molecule, the T cells can recognize the peptide presented through the TCR alpha and beta chain. Here there is the CD3 complex constituted of different chains that will transduce the signals. There is the binding site for the co-receptor of CD8. In here we have the target cells but now we have the class II molecule with alpha and beta chain, between the alpha one and the beta one we have the molecular pocket in which we have the peptide that is recognized by the TCR of the T cells. We have the CD3 complex of chains, in this case we have a class II and so we have a CD4 co-receptor. We have also these other T cells that has a gamma-delta chain receptor that is a minor population (5% of the lymphocytes) and these lymphocytes express CD3 and low CD4 or CD8. They are important because many features of these cells have to be assessed yet, but we know that their TCR directly binds the microbial antigens or stress antigen that are present in suffering cells. This gamma- delta T cells are present in the epithelia and in the gut mucosa and they are able to rapidly produce cytokine or kill pathogens. 16 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 INFO We have also invariant NKT cells, invariant because they have a TCR that is more or less always the same (no big variation unlikely the T cells). We can discriminate this population using again the CD3 marker but also some functional markers such as CD56 or CD16. This minor population expresses a peculiar TCR, that has a limited specificity, which also interact with CD1 membrane molecules and not with the HLA molecules. Therefore, their TCR is able to recognize CD1 molecules. These cells do not express CD4 or CD8 because we talk about CD56 or CD16 and share features of both T and NK cells. Pay attention to not confuse these cells with NK cells, because they have a TCR that is able to recognize bacteria lipids and glycolipid presented by CD1 molecules, which are molecules coded by genes that are outside the HLA gene cluster and they are related to class I and B HLA molecules. Remember that they can recognize through a CD1 presentation. Thanks to the ability to recognize the lipid antigen, these cells are very important because they can help us in recognizing bacteria and virus. They act basically by producing different cytokines, that are IL4, IL10 and interferon gamma; those cytokines can influence the T cell response towards a regulatory response or an effector response because depending on the environmental factors and on the presence of the most secreted cytokine we can have the enhance of the immune response, it will have the secretion of IL4 or interferon gamma; or we can have the inhibition of the immune response with the secretion mainly of IL10. NKT cells are related to tumors and autoimmune diseases. There are T cells associated with mucosae (MAIT=Mucosal Associated Invariant T cells) that are defined as unique innate-like T cells that are a bridge between the innate and adaptive immunity. These cells show a limited T cell antigen receptor (TCR) that is concerned across the species. Which are the functions of this kind of T cells? They are basically activated by microbial riboflavin-derivative antigens and they are presented by non- classical MHC class I molecules. They have an important role in the defense against bacterial infection, but also in different kind of non-infection diseases. B cells In this case we have several B cells subpopulation, the most common one is the follicular B cells, that are present in the blood and in the lymphoid tissues. They are important because they generate the majority of antibodies, with a high specificity for the antigen, and also are important for the memory of the cells. We have also the marginal zone B cells that is a minor population too, which produce antibodies with a limited diversity. We have the B1 cells that represents a minor population and produce antibodies with a limited diversity. They form mainly in the marginal zone of the spleen where we have marginal zone B cells, while the B1 cells are formed in the mucosae and pleural peritoneal cavities. 17 Daniele Friolotto/Matteo Magliano – Lezione 8 Immunology - Prof. Curcio (1) – 28/10/2021 The B1 cells come from the fetal liver hematopoietic stem cells, where we have the development of pro and pre-B cells and then of an immature B cell. Finally, we have this structure: on the membrane of B1 cells we have an IgM whose function is like a BCR, as a receptor, and a CD5. If we analyze the percentage of these cells, for example, we can use the CD5 marker, while the follicular B cells and the marginal zone B cells, came from the bone marrow hematopoietic stem cells, where we have the pro and pre B cells. These immature B cells will mature in the secondary lymphoid organs in follicular B cells, that have an IgM or an IgD on the surface as BCR, or in the B cells of the marginal zone, then we can have an immunoglobulin present into the surface of those cells and also these other to marker. Questions: Do they take igG and igM at the same time or they take either? Commonly either. Just in the first day after the birth you can have again some cells, but then they will go down (the B1 type) 18 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 Antigen Receptors of T and B cells (TCR and BCR) INTRODUCTION In the past we have talked about the general overview of the adaptive immune response and how our lymphocytes circulate in our body and so they are able to almost visit every day all our lymph nodes, thanks to the circulation through the blood and the lymphatic vessels. Which are the three main signals regulating this recirculation? 1. Chemokines, that are constitutively expressed in lymphoid organs, (we mentioned 2 chemochines that are bound by the unique receptor CCR7 that is expressed on naive lymphocytes. CCR7 is also expressed in dendritic cells that must travel from the perifery to the lymphoid organ to present the antigen to lymphocytes) 2. L-Selectin, that mediates not an actraction but an adhesion. It is highly expressed in naive but not in effector T cells, and binds on adhesion molecules on peculiar endothelial cells termed high endothelial vessel. 3. Sphingosin I Phosphate receptor, expressed on T cells, it mediates the recirculation, especially the exit from the lymphoid organs, otherwise the T cell has to stay there for some days. It is very important because when the virgin lymphocyte comes in the lymph node, (it has never encountered the antigen) re-expresses immediately this receptor, because of the low concentration of sphingosin I phosphate into the lymphoid organ. But if it encounters the antigen, it is activated and expresses the CD69, (that is a short marker activation, that appears and disappears in a couple of days) that is sufficient for lowering the expression of sphingosin I phosphate receptor. In this case the lymphocyte cannot go outside, because it cannot perceives the sphingosine I phosphate, that is abundant in the blood or lymph. This is important because allows the lymphocytes to be activated and to remain into the lymph node and proliferate and clonally expand, how they should do, to give the arm with the same specificity and able to go outside and reach the perifery where the infection is on going. These are the 3 main mechanisms by lymphocytes B and T. What is the difference between B and T in the recirculation? Only the chemochines, because even the B cells adhere to the high endothelial vein and response to chemokine, that is a different one. In an immunohistochemical immage of the lymph node we can see that the T cells are abundant in a specific area and the B cells in another one. This is possible thanks to the expression of different chemokines into different areas. The rest is similar, so even the B cells express the sphingosin I phosphate receptor and in this way after the diferentiation in plasma cells they can reach the bone marrow or the perifery. Today we will discuss the recombination, so the appearance and the generation of the antigen receptor of both. They are structured very differently, but the process, the somatic recombination, that generates those receptor is similar. We will try to parallel and discuss the differences between the 2 lymphocytes. 1 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 The T and B receptors belong to the Immunoglobuline superfamily, like the HLA class I and class II, some adhesion molecules, some markers specifically expressed in the immune cells (like CD4, CD8 and CD3) but not only. They all belong to this superfamily, characterized by the presence of many globular domains, that can be different in each molecule. So the TCR and the BCR are actually similar, even if different especially in size. This picture represents the TCR. It is made by 2 chains (blue chain=α, green chain=β), so it is an etherodimer. These chains are present in the majority of our lymphocytes (80-85% of circulating lymphocytes) and those are termed αβ lymphocytes. 2 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 But we have also small portions of lymphocytes, that are called γ-δ, because instead of having α and β chains they have γ and δ chains, but they are very similar. So, the δ and the β are similar, the same for α and γ. We will never see a different combination; we will never have α-delta or γ-β. Now we will see why. The TCR is made by a big extracellular portion, a transmembrane domain and a very short intracellular portion, indeed is not able to transduce the signals by itself, but it has to be coupled with the CD3. CD3 is a huge molecular complex composed by at least six different chains, that interacts with α and β chain of the receptor. When the receptor binds the specific antigen the change in conformation of the receptor will be perceived by the chains of the CD3, binding the receptor, and inside will start the phosphorilation cascade of the ITAM domain (Also the CD3 has an ITAM domain in the cytoplasmic portion). This will allow the transduction of the signal. BCR The BCR is a heterotethramer, because is made by 2 heavy chains (green) and 2 light chains (blue). The 2 heavy chains are identical and the 2 light chains too, but they are coded by different genes, so they are independently sinthetized. Even the BCR is made mainly by the extracellular portion (that can differ in the number of globular domains), a transmembrane portion, and a short cytoplasmic tail. Even the BCR is not able to transduce signals by itself, but it needs the association with the co-receptor for the BCR (it’s not CD3, that is a marker only for T cells). Co-receptor for BCR is made by 2 others very short immunoglobulines, called Igα and Igβ, that again contain in the intracelluar portion the ITAM domain, and so with the changing conformation of the BCR binding the specific antigens, it is perceived by the co-receptor Igα and β that will start the signal cascade. 3 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 We have already discussed the generation of the huge repertoir that characterizes the mammalians and in particular the humans. It is generated by a precursor and in a specific moment, during the maturation of T and B cells. After the maturation they will never change the receptor that expressed on the cell surface. The appearance of the receptor exposed on the cell surface, both on T and B cells occurs without knowing the antigen, because occurs in absence of antigen. So the big question is: how it is possible that we have so many T and B cells able to potentially recognize even synthetic peptides (so antigen that are not present in nature)? If we reason 1 gene= 1 protein (as in many cases, of course) we should have billion of genes each to give so many different TCR or BCR, instead no, and we will see why this. Before to see the gene structure of TCR and BCR, we see some details about the protein structure, because it is important to understand it after the recombination. This is the α-β TCR. We have to go deep in detail, because each receptor and each chain making the receptor is actually constituted by a Constant portion, that is called C (divided in Cα for α chain and Cβ for β chain) and a Variable portion (V), again one for the α (Vα) and one for the β (Vβ) chain. The variable portion binds the antigen, and actually the costant portion is similar for every TCR, so every β or α chain has the same costant portion, but what is different from lymphocyte to lymphocyte is the variable portion. In the variable portion, we can distinguish other portions that are called hyper- variable portion, or CDR (Complementary Determining Region) and we have 3 of these CDR (1, 2, 3) in each variable portion. This is very important especially the for the CDR 3 that is binding the antigen. They are thus called “complementary determining region” because the CDR are those determining the complementarity between the TCR and the peptide HLA complex. In particular we will see that CDR3 is the one binding the antigen, while the CDR1 and CDR2 are the ones determining the complementary binding with the HLA molecule. For this, the TCR is actually recognizing not only the peptide but also HLA molecule. 4 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 The same is for BCR. Each heavy or light chain is made of a variable portion and a constant portion. The constant is again similar to all BCR and expressed by all lymphocytes, while the variable portion is different. The variable portion is the one that binds the antigen. What’s the big difference between the TCR and BCR? The BCR has 2 binding domains for the antigen, while the TCR has only one. In the BCR, however, these 2 antigen binding sites are the same, because the 2 light chains are the same and the 2 heavy chains too. So, the two variable region in the same receptor are the same. And again, even for the BCR, in the variable region we distinguish 3 CDRs, that are responsible for the complementarity of the antigen binding site and the antigen. We have CDR1, 2 and 3, that are the hypervariable region. What does “hypervariable region” mean? It means “more variable than the variable”, so means that even if 2 lymphocytes can have and can use the same variable segment to make variable regions, we have a mechanism that will create some hypervariable regions, and so these 2 lymphocytes can actually bind similar antigens, but with different affinity or even a different antigen by the mean of a mutation of only few nucleotides. 5 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 How is it possible that 2x104 genes present into the human genome code for more than 10 11 different TCR and BCR? This is possible thanks 2 different somatic strategies (they are called “somatic” because they occur in the precursor T and B cells, so they are not in the germinal line): 1. Random recombination of multiple gene sequences present in our genome: it is a recombination of different DNA segments that constitute the genes of TCR and BCR 2. Small errors during the recombination process This is the structure of the genes. For TCR we have the α or γ, that are similar, an the β or δ, too. All these regions are actually involved in making the variable region or the constant region. Each α or γ chain is composed by a variable region made of many segments, called variable DNA segments, and many segments called joining segments, while in β or δ chains we have also other segment between variable and joining, called diversity segment. The same is for the BCR chain, so the light chain that is very similar in the structure to the α chain, so the shorter one, made by different DNA segments, and different joining segments. The heavy chain is more similar to the β chain of the TCR, and it is made again of different DNA segments, called variable, and different joining segment. The constant portion is similar for each α, β, light or heavy chain, and it is made of unic DNA segment, the constant one, that is not actually only one for each chain but very few in comparison with variable or joining segment, and then the transmembrane portion and the cytosolic tail. 6 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 Here is the protein structure, with the constant portion, transmembrane and cytosolic tail, that is actually coded by a unic exon, and for the β chain, the variable region, constituted by a segment from the variable gene, a segment from the diversity, and a segment from the joining. The same is for the α chain: the variable region is made of one segment coming from all the segments called variable, and one from the joining segment. This is the β chain, made of variable diversity, joining segments. We can see here that the variable region of the β chain is made of more than 50 different DNA segments, following each other. The diversity region is made of 2 different segments, the joining region is made of 6 and 7 different segments (a dozen in total). It happens that actually we have to join together to make an exon that contains only one of these 50, one of these 2, and one of these dozens of segments. How is it possible? Through the recombination, that for this reason is called V-D-J recombination. 7 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 Here is the structure of the δ chain, very similar to the β one, so it is bigger, again with a variable, a diversity and a joining region, plus the constant portion. Here we can see that the variable is actually made of few segments (2 or 3), and the diversity is made of 3 segments, the joining 4 segments. For this reason, we say that in the γ-δ receptor there is less diversity in the receptor. In the past time we saw that the γ-δ lymphocytes have less diverse receptors in comparison to α-β. This is the reason: we have less DNA segments among wich we can chose and generate the receptor. We can also see the gene structure of α chain. This is made of variable region, in which there are more than 45 DNA segments, a joining region, with more than 50 segments, and then the constant portion. It is important to note that the δ segments are inside the α chain, so the first event to generate the TCR is the recombination of the β chain. We don’t actually know why the β chain, it is actually a matter of how the chromatine is opened, and usually for the T cell precursor the signal coming from the stromal cells, but also the epithelial thymic cells, is inducing the opening of the chromatin in the β region (they are different chromosomes). The β chain is the first that recombine and if it works correctly and is structurally perfect, the thymocytes will start to recombine the α one, that means that it’s time to choose one of the variable segments that need to be joined with the one of the joining segments and all the DNA segments in the middle (including the δ region) will be cut away and degradated. For this reason, is not possible to have a β chain paired with a δ chain. 8 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 This is the γ chain, that is like the α one, so again we can notice the lower number of DNA segments in the variable region or in the joining region. A similar structure is also for the heavy and light chain of the BCR. We have again more than 45 segments in the variable region, more than 20 diverse segments and 6 or 7 joining segments. The same is for the light chain, like for the α chain of TCR, in which we don’t have the diversity segments, but we have a lot of variable segments. We have 2 different light chains, the κ or the λ chain, and so each of these has the same structure. There are many segments again for the variable region (more than 30) and for the joining region (7) and the same for the λ. Then we have all the constant part. In the case of BCR the constant part is a bit bigger than the TCR one. This because we can have actually different isotype of antibodies: IgG, IgM, IgA, IgE, IgD. These are different for a different constant portion, and so the gene coding for the heavy chain needs to have a different segment also for the constant portion. 9 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 Now we have to understand how the recombination is giving only a simple RNA, coding for the β or α chain with a very simple variable region and consant region, for the α and for the β chain. The same is for the heavy and light chain. The rearrangement or recombination is crucial for the generation of this receptor, and it is a process that occurs only in B and T cells, specially in T and B cells when they are still immature. How does the recombination happen? It is called V-D-J recombination because the goal is to join together one of the segment of the variable region with one of the segment of diversity region and with one of the segment of the joining region. We saw that they have many DNA segments, but in the end each cell needs to have a single and easy RNA coding to be transduced in a single chain. This happens thanks to a site-specific recombination. It is important to understand that with this recombination process all the DNA that is between the 2 segmetns chosen to be joined, is lost forever. Let’s see step by step this process. We have here again the genetic region of the β chain (V, D, J). The first step is the joining of one diversity segment with one joining segment. They are randomly chosen. The 2 segments become very close, thanks to some molecules, and all the DNA in the middle is actually making a ring that will be detouched as an episome and will be also degraded to recover all the nucleotides. So we have the 2 first segments joined. Then we have to chose again randomly a segment from the variable region and this will be joined with this complex (D and J). And again, this complex will be closed by 2 variable segments, thanks to the cooperation of other proteins, and all the DNA in the middle will be again lost as an episome and be degraded. So in this way we have the V-D-J portion that will code for the variable region of β chain, that can be actually not so close with the constant portion. 10 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 The primary RNA is not the final one. The mature RNA is the one that, through the alternative splicing, is giving the segment V-D-J, just rearranged, in proximity with the constant portion. This will be the RNA that will be translated in the β chain. The first step of recombination in T cells is the recombination of β chain, in B cells is the recombination of heavy chains. So the first step of V-D-J recombination is the joining of one D segment with the J segment and then the joining of this D-J with one variable segment. And then we have again, through the alternative splicing, the proximity of this variable region with the segment coding for the constant portion. We have now a very easy RNA, starting from a lot of segments. So, V-D-J recombination is: Tissue specific, because it occurs only in B and T cells Timely regulated, because it occurs only when they are immature, so when the T and B precursor have to generate the fully differentiated T and B cells Cell cycle phase specific: it occurs only durig G 0-G1 phase so when they are not proliferating, because it is important that during the recombination, they are not duplicating the DNA. It is important for a T cell to have a single TCR Characterized by the allelic exclusion Allelic exclusion Whenever the recombination starts, it starts only on one of the two alleles, chosen randomly. If it is successfull (V-D-J recombination and the β chain or heavy chain are structurally ok) the recombination is not occurring on the other allele. This happens because the β chain is exposed on the cell surface of the pre-T and pre-B cells. Thanks to the combination with a surrogate complementary chain (so for a beta chain will be a surrogate alfa chain, for the heavy chain will be a surrogate light chain), it is checked on the cell surface for the binding and the affinity and the functionality of the antigen binding site. 11 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 If everything is ok, the other allele coding for the segment gene for the β or heavy chain, will be excluded. So it means that the recombination stops there for β and heavy chain, but it will start of course for the complementary chain, so the alfa one for the TCR or the light chain for the BCR. It is also important that there are 2 enzymes, called RAG1 and RAG2 (=Recombination Activity Genes) that are specifically expressed on the pre-T and pre-B cells, and they are not more expressed when the lymphocytes are mature and circulate in the perifery. So the expression of these genes is typical in primary lymphoid organs, so the immature T and B cells in bone marrow or in the thymus. How is it possible that we have so many different TCR or BCR? If we consider α-β, we have for the α chain 75 variable segments tha can be randomly joined with 61 joining segments. So if we calculate the probability, we can have 75V x 61J = 4574 of different potential combinations For the beta chain we have 25V x 2D x 12J=600 different combination of binding sites. Then in each cell there is still the random combination of one α chain with the β chain, so: 4575α x 1200β = 5,5 x 106 different combinations. So thanks to the germ line inheritance we can have 5 millions of different αβ TCR The same is for BCR: in the light chain we have 38V x 5J (κ) + 33V x 7J (λ)=421 different binding sites. In the heavy chain we have 46V x 23D x 6J=6348 different binding sites. Again, the random combination of the heavy chain with the light chain, so we have 6348H x 421L = 2,7 x 106 different combinations. 12 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 Actually, if we see in total, we cannot reach what we really have in circulation, so this means that through the recombination we are having more or less 7/8 millions of different TCR and BCR, but we have still to reach huge number. How is it possible? Thanks the other strategy that we anticipated that is called junctional diversity. It is actually what is generating specially hypervariable region in the variable region, so this means that this V-D-J recombination is not so accurate, so some mistakes can occur, and these mistakes are not so bad, because they can generate the structure that is not working, so lymphocytes will die, because they are not functional, but can be also successful and give a higher affinity of that receptor for the antigen. So, this variation strongly increases the variability and everythig is drove by these RAG proteins. Now we will see in detail step by step what happen during this recombination and specially during the joining that is allowing this huge diversity. So the RAG genes are actually recognizing specific region in the DNA (because they have to put together a segment from the joining region and then from the variable region to the diversity) so it’s necessary that they recognize the Recombination Signal Sequences (RSS). These are specific sequences made by an heptamer and a nonamer, that are similar in all our cells, they are specific for T and B (in which they are the same) and mantained in the human being. The heptamer and nonamer are both palindromic, so they need to be complementary, and between the heptamer and the nonamer there are some spacers, that can be of 2 types, 12 or 23 nucleotides, that are not conserved filogenetically, so they can be different. The 2 conserved sequences are only the heptamer and the nonamer. So we have heptamer, 12 nucleotides and nonamer, or hepatamer, 23 nucleotides and the nonamer. These are the 2 potential RSS recognized by the RAG protein. The RSS for the D segments are down and up each segment, on the 3’ and 5’, and actually the RSS are only on 5’ of the joining segment and on the 3’ of the variable segment, because the diversity segment is joining with the joining segment and then is joined with the variable segment. The possible combination, the only one, is the combination of 2 different spacers, so for this reason is actually said that the V-D-J recombination is following the 12-23 rule. If the heptamer and the nonamer should be aligned, because they are complementary, this means that one of the two strands should be actually longer, because it has to turn around the other one, to align heptamer and nonamer. The 2 spacers are different because one of the 2 has to make a turn to the other strand to allow the combination of heptamer and nonamer sequence. So for this reason the same spacer with the same lenght is not able to make a turn. So the heptamers are aligned and the longer one is making a turn around the other space in order to have aligned also the nonamer, and the RAG that are recognizing all the sequences have also the nuclease activity, so can actually cut the DNA very close to the 13 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 diversity and joining segments and then very close to variable and diversity segments when they are engaged in the other recombination. It happens that all the DNA that is in the middle, that contains the segments that are not chosen, goes away through the episome formation and degradation. What happens to our selected segments? There are different ends, but because of the double strand break is a huge damage perceived by our cell and the chemical structure of DNA these 2 ends are immediately chemically joined and closed just making a hairpin structure. 14 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 This doesn’t repair the double strand break, but because of the oxydril on the phosphate on the other group they are closed. So needs that another enzyme is involved in this process, called artemis. This endonuclease is cutting the DNA, to open the double strand, but randomly again. If we look at the sequence we have A-G-C-T. If artemis is cutting here (the yellow arrow on the left in the first image) because of C and G are joined means that A and G will go up. The same if is cutting here (second arrow), before A and T: on the other strand these 2 nucleotides wil go down. Artemis is also able to generate some new nucleotides on one strand, that were no present before, and these nucleotides are called P nucleotides. Then, we have to join this, because we have to repair this double strand break, so there will be a polimerase that just for the complementary rule is filling the gap. But can also be involved another enzyme, that is called TdT (Terminal desossinucleotidil Transferase), that is able to add other nucleotides, without following a template, different from a polymerase that use a template. In this case this particular enzyme, that is expressed only in the immature lymphocytes is adding (in blue) random nucleotides, with no rules. Because of these are put randomly, they are not always complementary, so it’s necessary that some endonuclease will cut the one in excess, in order to have a complementary terminal. 15 Matteo Magliano/Marco Sanfilippo – Lezione 9 – Immunology (Prof. Cappello) – 02/11/2021 In this way we have the polimerase that is filling the gap. So the pink nucleotides added by the polimerase are complementary to the ones added by the TdT, filling the gap and closing the break. So, from the first 2 nucleotides we have at the beginning where the RAG was cutting (TC and TA) we have a lot more nucleotides. This is what is responsible for hypervariable region, so for the CDR region. This is what is actually increasing the different repertoir we have. Indeed based on germ line we could have actually 7/8 million of different TCR, but thanks to this somatic recombination and thanks to joining variability we can reach a hundred billion of different combinations. Then we can have actually much more, because if we do the sum of these 2 strategies we should have 1017 different BCR and TCR, but actually we only have hundred billion, because many of receptors just generated are not functional, and the lymphocytes are just dying because not functional, or not having a functional receptor which is the only molecule able to really transduce the survival and proliferating signals to the lymphocytes. So, the circulating ones are a huge number but not as much as we could have. 16