Immunology Lesson 13, 14 PDF

Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 Summary and continuation of the previous lesson CTL (CD8 cytotoxic T cells) can directly kill infected cells like cells infected by intracellular bacteria, tumour cells, tissue damage in the presence...

Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 Summary and continuation of the previous lesson CTL (CD8 cytotoxic T cells) can directly kill infected cells like cells infected by intracellular bacteria, tumour cells, tissue damage in the presence of some viral infections. The signals that mediate their fully differentiation are: IL-2 IL-12, IFN-γ and IFN-1. These cytokine signals are important because they induce the expression of the master regulator genes and so the transcriptional factors specifically expressed by cytotoxic cells: Tbet and Eomes. These two transcriptional factors will induce than the expression of perforin and granzyme B. Instead, T helper are important because they recognise the peptide present in the complex with HLA class II and that means that they are mainly involved in the response against bacteria, especially the extracellular one but they can also be induced from intracellular bacteria thanks to the ability of dendritic cells. There are different Th subsets which are all characterised by the release of different types of cytokines. In fact, Th produce cytokines and this is important because the cytokines induce the activation of different immune cells. T helper are also important because they are mandatory for the differentiation of CTL. Each sub-population of T helper cells can activate different kind of cells. In the past lesson, we discussed also about the tricky role of Th 17 because, together with Th 1, in some particular conditions, they can be pathologic because they can induce the damage of our tissues and be responsible of the appearance of some autoimmune disease. Now we can speak about the Tfh. Tfh (T follicular helper cells) The follicular helper cells are those T cells that, after their activation, remained trapped in the follicle and so in the lymphoid organ and they can acquire actually different profiles: Th1, Th2, but sometimes they can also release IL-17 and so Th 17. They are actually like Th1, Th2 Th 17, so all the other subpopulations, but they express the Chemokine receptor that maintains those T-cell in the lymphoid organ. Their remaining in the lymphoid organ is very important to support the B- cell activation. Among the signals that mediate their differentiation there are: IL-6 and ICOS-L. The ICOS-Ligand expressed by the antigen presenting cells bind the ICOS Receptor (expressed by T-cells). This binding is important because it maintains the expression of the CCR7, that is the receptor that mediates the circulation of naive T cells, into the lymphoid organs. In fact, it binds the chemokine expressed in T cell area and induces the expression of another chemokine receptor that binds the chemokine constitutively expressed in the B cell area. Therefore, these particular cells can actually migrate into the lymphoid organs between the T cell area and the B cell area. 1 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 This is important because they are activated as usually in the T cell area by the contact with the antigen presenting cells, mediated by the biding of ICOS-L that induce the expression of CCR5 that mediates the travelling into the B cell area in which in the meanwhile, the B cells are activated by the antigen itself. So, they are important for the B cell activation. Through the secretion of cytokines, they will influence the isotypic switch. In fact, as we know, we have different types of antibodies but all mature B cells in the periphery express only IgM and eventually IgD. So, if they are not able to make the isotypic switch, they will produce forever IgM or eventually IgD. Thanks to the cytokines, released by Tfh, they can change the isotype and so also express IgA, IgG or IgE. The naive CD4 T cells are usually activated with all the signals that we know and thanks to the ICOS activation, they will express the Chemokine receptor that will leave these helper T cells to be contacted from the B cells. These cells express ICOS-L and so trough the ICOS they fully differentiate in Tfh cells. So, they remain trapped and will produce the cytokines, which are important for the isotypic switch. Before that, they will produce the IL-21 that is very important, like the IL-2 for the T cells. In fact, it maintains the proliferation of B cells activated by the recognition of the antigen. They support the proliferation and all the steps that we will see. This is what happens into the lymphoid organ and we know that all the other T cells subsets, including also the CTL, are going away to reach the periphery. On the other hand, the B cells need to be still activated and do not actually leave (or eventually only the memory B cells leave the lymphoid organ) because they need to differentiate in plasma cells to reach or the bone marrow or the lymphoid organ associated to the mucosa. Actually, only the plasma cells leave the lymphoid organ. The Tfh are those that eventually migrated again in the T cells area when they need to be reactivated. 2 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 Indeed, you can see, if you stain the usual section of a lymph node for the CD3 that many T cells can be in the B cells area when the foreign antigen has been presented to the T cell and eventually it is recognised by the B cells. This video is a summary of T helper subsets differentiation: https://www.youtube.com/watch?v=Qs1H5P0SaLU B CELL ACTIVATION The spleen The spleen is the major secondary lymphoid organ (the primary lymphoid organs are thymus and bone marrow) for the size and for the number of cells that it can hosts, especially B cells, in fact more than 60% of cells in spleen are B cells. It has a complex structure and it’s rich in blood vessels as the splenic artery and the splenic sinusoids. The spleen is divided in two compartments: red pulp and white pulp. The red pulp is mainly composed by fibroblasts, reticular cells, connective cells. This is the area in which:  old red blood cells are removed  immune complexes are destroyed  old and damaged lymphoid cells are removed The red pulp has also a storage of platelets because there are a lot of megakaryocytes. The white pulp has a similar structure to lymph node because it is constituted by a T cells area, B cells area and especially it is divided in trabeculae so there is a cortical part. In this area there is the presentation of the antigen by the antigen presenting cells to the T cells and the activation of B cells. 3 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 We can see similar structure to lymph node and similar to the white pulp of the spleen in lymphoid tissues associated to the mucosa. In fact, we can distinguish a B cells area and a T cells area. In the image you can see the follicle and all around the T and B cells but also some plasma cells that differentiate there and antigen presenting cells. These lymphoid tissues usually associated to different types of mucosae are not well separated by the others tissues. So, they don’t have trabeculae or capsule around the lymphoid organ. In this case in which we don’t have all the complex organization of the lymphoid vessels and blood vessels, the antigen arrives inside thanks to particular cells (especially in the GALT – gut associated lymphoid tissues) called M cells that are important to allow the antigen present outside to be transported inside, closed to the lymphoid organ, ready to be presented by the antigen presenting cells or being recognised by the B cells. M cells (Microfold cells) M cells are located in the intestinal epithelia closed to lymphoid follicles, near the Peyer patch or other GALT structures. The M cells are very important because:  They allow the passage of the antigen from outside to the inside without any damage in the anatomical barrier.  They can capture the antigen and even the whole bacteria from the intestinal lumen, thanks to their short microvilli and large gaps.  Thanks to the ability of transcytosis of microbial antigens they will invaginate the membrane. So, the M cells trough the microvilli will bind the microbe, will invaginate the membrane, will make the endosome that will be released outside in the lymphoid tissue associated to the mucosa (without of any kind of degradation). Then it can be recognised by the B cells or by the antigen presenting cells (especially dendritic cells) and then processed and presented to the T cells. The M cells then allow the transportation of the bacteria from the lumen to the inside of the tissue, in order to induce the adaptive response. Some bacteria like the Salmonella are not recognised by the M cells so they are not transported in a controlled way and they are cytotoxic for our GALT, allowing the entrance of other microbes. So, in this way we lose the controlled transport of the microbe inside the tissue. Because we have lymphoid vessels everywhere like for the blood vessels, microbes that enter through the Microfold cells in MALT or GALT can reach the lymph node or spleen and induce a greater adaptive response. 4 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 These cells are also called “window” because they interrupt the ciliate epithelium of GALT because they have short microvilli that not produce the mucus and so the bacteria are not trapped and so they can be endocytosed and then released in the other side. T- dependent and T- independent B cell activation Talking about B cell activation, we have to distinguish 2 two different kinds of activation. This differentiation is mainly based on different types of antigens. We can distinguish a T-dependent B cell activation and a T- independent B cell activation. In the first case the T helper cells are mandatory for the activation of the B cells, while in the second case the activation is mediated by the presence of the antigen and eventually by some other stimuli like the presence of complement but not by the T cells. In these two processes there are many differences:  The first difference is the type of B cells that undergoes to this kind of activation. The T- dependent activation is typical of B2 follicular cells so the one that are into the follicles into the lymphoid organ. The T- independent activation is typical of B1 cells or MZ B cells.  The second difference is the type of antigen. In T- dependent activation we have a protein antigen. In the other case, (T-independent) the antigen is a polysaccharide, so all the sugar 5 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 that are present on the membrane wall of the bacteria, or even on the viral capsids, can be recognised by the B cells and this is enough to activate them and to produce the antibodies  The antibodies produced after these two different mechanisms are different. The T- dependent B cell activation will produce at the end different kind of antibodies: IgE if it has to fight against big parasites or IgA or IgG. The antibodies produced in response to T- independent antigen are always IgM. In very rare occasions can be IgA.  Also, the type of plasma cells producing these antibodies is different. Plasma cells deriving from B cells activated from T-dependent antigens are long live plasma cells that means that they can survive for months in our bone marrow and they can produce for some weeks the same antibodies. Plasma cells deriving from B cells activated from T-independent are called short live plasma cells because they remain in the lymphoid organ for short time, just a couple of weeks and they produce only IgM.  The last important difference is that only after a recognition of a T-dependent antigen, the B cells differentiate in plasma cells and some of them will also differentiate in memory B cells. This process doesn’t’ happen with the recognition of a T- independent antigen. It’s important to remember that the T-dependent antigen is always a protein because it needs to activate also the T cells and this means that the B cell is helped in its activation by helper T cells and they will differentiate in memory B cells and long live plasma cells, producing different types of antibodies. While the T-independent antigen are always sugar or lipidic molecules that are not recognised at all by the T cells and so they will induce the differentiation in short live plasma cells, that will produce only IgM. T-dependent cell activation It involves only the follicular B2 cells. The antigen arrives in the lymphoid organ through the antigen presenting cells, it will be activating T cell and it will differentiate in T follicular cells. These cells will help B2 cells that will recognise the antigen and will start a short proliferation and then, thanks to the help of the Follicular T cell, a huge proliferation occurs. All these processes of proliferation are included in what we call the Germinal Centre reaction. So, instead to be disperse in the B cell area, those activated by the recognition of the antigen will start to proliferate and make a very small clone, but when they receive the help of follicular helper cells (that induce the IL-21) a huge proliferation and expansion will start, creating the germinal centre. 6 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 The germinal centre is divided in two areas: the light area and the dark area. This is important because it is used by the medical doctor to make some diagnosis. For example, people that is lacking in some of the costimulatory molecules, important for the fully activation of B cells, don’t have the germinal centre, and so this is important to make a diagnosis of immunodeficiency. As we know, these people will never have memory B cells or different isotypes of antibodies, they will always produce IgM. The germinal centre is also called secondary follicle and this is made by clone of B cells that are activated by the antigen, only and always after the help of T cells. The B cells on the surface, as many copies of BCR, can recognise the antigen and enter through the lymphoid vessel or blood vessels. The recognition of the antigen induces the cross linking, so the recruitment of more copies of BCR in a very small area on the surface of the B cells. This implies also the cytoskeletal reorganization and induce the gene activation that means that B cells, as we saw for T cells, after the activation need to express new genes, for example the kinase cyclin dependent to allow the proliferation. In this particular case the B cells need to rearrange the cytoskeletal quite strongly because they endocytose the complex BCR antigen. As you can see here, the antigen bound to the BCR is endocytosed by the B cells and like a normal antigen presenting cells, it is processed and then presented in association with MHC class II molecules. 7 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 As we know, when the antigen is phagocytosed by the external environment, like even the antigen complex BCR, is contained into a vesicle that then will fuse with the lysosome and so the antigen end even the BCR will be degraded. After the fusion with the post Golgi vesicle, in which the MHC class II molecules are brought to the surface, this antigen can be loaded to MHC class II molecule and then exposed to the surface. The B cells can present the antigen loaded in the pocket of the MHC class II molecules. This is important to make a strong and long-lasting contact with the T follicular cells that have been pre- activated by the antigen presenting cells. So, the T follicular cells have already seen the antigen but when they encounter the B cells, that expose on the surface the same antigen loaded in the MCH class II, can again be engaged through the TCR because the TCR is specific for this complex. At this point the B cell is activated again, so it boosts the T cells and in consequence the T cells will release cytokines and other signals necessary for the B cells activation. Then the activated B cells will start the huge proliferation and will give a clone, which then differentiate in plasma cell and will produce the antibodies with different affinity for the same antigen. CD40L and CD40 All this is possible thanks to the biding of two specific molecules: the CD40 Ligand that is present on the T cells and the CD40 that is present on the B cells. This interaction is very important because it induces all these processes: - It’s important for the B cell because the CD40 bound by the CD40L induces the survival signals to the B cell, so it induces the expression of the antiapoptotic molecules - It induces all the germinal centre reactions - It allows the production of different kind of antibodies (isotype antibodies and so the isotype switch) - It induces the most important reaction that we call affinity maturation of the BCR. 8 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 All this is possible thanks to the expression of a specific enzyme called AID (Activation-Induced Deaminase) that is induced specifically by the signal of the CD40 in B cells. In addition, CD40 allows the differentiation of memory B cells. It’s important to remember that the lacking of one of the two signals (CD40L or CD40), gives an immunodeficiency phenotype: without the germinal centre, they have not memory B cells and they have not different kind of antibodies so they only produce IgM. This deficiency is called Iper IgM. Because of this lack we have also a less efficient T cell activation because the CD40L is also important to induce the co-stimulatory molecules that are important to give the second signals to the naive T cells. What happens in the germinal centre? In the light area of the germinal centre, we have the centrocytes while in the dark area we have centroblasts. These are two differentiating statuses of activating B cells. In the germinal centre there are 3 important processes: 1) Hypermutations: it occurs in the dark area, only in the centroblasts when the B cells are proliferating 2) Affinity maturation: it occurs in the light area, in the centrocytes. This process is important because allows the survival of only those B cells that produce the high affinity antibodies for the antigen. 3) Isotype switch: this is a process that changes the class of the antibodies produced by the differentiated plasma cells. Centroblasts and centrucytes are different because the centroblasts are essentially with the B cells that are proliferating and so all the content is nucleus and at the histological staining they appear dark, while the centrocytes are those cells that exit from the cell cycle and express new BCR. We say new BCR because the first reaction that occurs after the germinal centre is the reaction in which the centroblasts and centrocytes can move in the dark area and in the light area, which is the Hypermutation. The Hypermutation is allowed thanks to the expression of the enzyme AID (Activation Induced Deaminase) which deaminates cytosine to uracil. In the centroblast that are proliferating, happens that this enzyme, which is expressed only in this particular state of differentiation, interacts with the single strand DNA and deaminates randomly the cytosine in the DNA strand. The presence of uridine that is not usual for the DNA and it is perceived like an error by the centroblasts and so it induces the DNA repair mechanism typical of our cells. The uridine can be excised as single nucleotide: this process is called MISMATCH REPAIR, or the uridine can be excised by the DNA strand with the adjacent nucleotide: in this case we talk about BASE EXCISION REPAIR. Of course, then the DNA needs to be repaired by the DNA polymerase that will fill the gaps made by the AID. 9 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 When the centroblasts are transcribing particular genes necessary for the division we have the two DNA strand separated. One that is coupled with the RNA is safe, because the ADI can only attack the single strand of the DNA. Where there are the cytidines, the ADI is just deaminating and transforming the cytidine in uridine. The gaps left on the DNA are then filled by the polymerase. For sure, the sequence of this strand will change and this kind of Hypermutation, that is somatic because it does not occur in the germ line, is actually occurring with a faster raid than usual. So usually, all our cells can undergo to somatic mutations, usually one base out of million bases. In this case in the activating B cells this occurs one base out of thousands, far more than usually. If you remember how is the variable region of BCR so the one made by DDJ segment, all that sequence is about 700 bases. So, it means that at any cell cycle the B cell undergoes to one hypermutation per cycle in that area. We saw that centroblasts can become centrocyte and then centroblasts again and so on. So, they can accumulate different mutations. They continue to accumulate different mutations even in the secondary or third response to the same antigen. If they accumulate different nucleotides, they will have different variable region. This can result in a higher affinity variable region but also a lower affinity variable region. And so, it means that when we have again to check this BCR. It is not like the check point during the maturation. In this case the BCR is more like the “mother of competition” because all the centrocytes in the light area that express a slightly different BCR will compete with all the others to receive the survival signal through the BCR that is given by the antigen. Only the one that will express the new BCR with higher affinity can win this competition, recognise the antigen and receive the antiapoptotic signal. It’s not a problem that BCR is not working, there is no problem because only one base cannot actually result in a completely different BCR. It’s a matter of affinity to the antigen since we need, to fight any kind of pathogen, the antibody that can be more affine than possible, because we know that if the antibody has a very high affinity with the antigen, we need only few concentrations of antibody to find the antigen. 10 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 How is possible that they are selected for the one expressing the higher affinity BCR? Thanks to these particular cells that are called follicular dendritic cells, present in the light zone of the germinal centre that are called follicular dendritic cells for their morphology (because they have long dendrites), but they are not deriving from myeloid cells like dendritic cells. These long dendrites can interact with many cells simultaneously, so with many centrocytes at the same time. They can present the antigen because in the dendrites, they present this particular structure that is called Iccosome that is made by a membrane vesicle, on which there are a lot of receptors for the antibody and for the complement. They can bind immunocomplex in particular the one made by the IgM that binds the antigens or can be bound by the complement component. Therefore, the Iccosome that express the receptor for the antibodies can recognise the complex through the antibodies or through the complement receptor they can also recognise the complex through the complement bound to the complex. Anyway, they express on the surface the antigen that was inducing the activation of the centroblast and centrocytes. In this way they can be contact by the centrocytes that will prove the affinity of the new BCR. So only the centrocytes that will have very high affinity BCR, will receive survival signals. All the other ones that are not able to compete because their affinity is lower, will undergo to apoptosis and then will be eliminated by the macrophages. At this point, after this first selection, the centrocytes have two choices: the first is again enter in the cell cycle and become again a centroblast; the second is to differentiate in plasma cells and undergo to the isotype switch. This figure summarizes all the travel of a specific BCR. The BCR that recognised the antigen makes contact with the T cell and starts with the germinal centre reaction and so the proliferation first. So, we have the centroblasts hypermutation process. Then it becomes a centrocyte and it goes to the light zone where it competes for the antigen biding: this is what is called affinity maturation, because all the centrocytes with high affinity BCR survive and then become again a centroblast or differentiate in plasma cells. 11 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 Isotype Switch The isotype switch is a process in which AID is the main enzyme responsible but the choice of the isotype switch is due to the cytokines present into the environment. So, the type of cytokine produced by the follicular helper cell gives the signal. In the figure we can see the structure of immunoglobulin genes. We can see also the VDJ, already recombined and all the constant portion; µ, ἐ, γ, α, and so on. Before all these constant segments, we have a sequence called S that is a signal sequence that is specifically recognised by molecules driven by the cytokines. In this case we have IgE production. The cytokine inducing the IgE is the IL-4. The follicular helper cells in the environment make IL-4 that recruit some molecules that bind the DNA in the sequence before the constant portion ἐ (epsilon) and before the µ (if the BRC is an IgM). Then is similar but no RAGs (recombination activating genes) are implicated to what the RAGs are doing during the recombination  they just make closed the two segments: the VDJ, the signal sequence before the µ or before the δ and the signal sequence before the chosen segment. Again, the AID here makes the break that will induce, in this case, the base excision repair and so it cuts away all the DNA that is included between. Then a ligase is joining the VDJ with the new constant segment. What is important to remember is that the cytokines present in the environment, produced by follicular helper cells, are the one recruiting the different molecules binding different signals sequence. For example, if we need IgG and so it means that we need to fight intracellular and extracellular bacteria or viruses, the follicular helper cells will produce IFN-γ. The IFN-γ recruits proteins that bind signal sequences before the γ. So, it will happen the same thing: the constant portion γ is making closer with the VDJ and the DNA in between is cut away. Then a ligase makes the final DNA segment with the VDJ closer to Sγ. Then after the normal alternative splicing, we have the mature mRNA in which there are not introns (all the no-translating sequences) and at the end, it will be translated in different antibodies with the same variable region. So, the affinity for the antigen is similar to the previous one but has different constant portion and so it will mediate different mechanism to fight the pathogen. 12 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 Here you can see all the constant segments that B cell can have. Like humans we have 4 different kind of IgG: IG1, IG2, IG3, IG4. So, we have 4 different γ segments. We have 2 different IgA so we have 2 different α segments and IgE. When expands itself, (this is the first step of expansion: the centroblast) and after the affinity maturation, based on the cytokine presents, the naive B cell that has an IgM can decide to recombine and produce IgA or IgG and in a second time, based on different kind of cytokine present, the memory IgE can decide to recombine the IgA. This is possible of course only if the segment has not been cut away from the memory B cells. So if it decide to become an IgG than it can choose between a lot of constant segments. If it becomes immediately an IgE which is the last one and this means that it will never change the isotype again. This is the summary of what is happening to the B cells. Starting from the dark zone the B cells will proliferate thanks to the IL-21, produced by the follicular helper cells, and during the proliferation it occurs the somatic Hypermutation. So, the centroblast that will exit from the cell-cycle will enter in the light zone and will express a different receptor, because after the hypermutation it will be slightly different and so this B cell will compete with all the others centrocytes to recognise the antigen presented from the follicular dendritic cell. If the new BCR has a lower affinity than before, they will never win the competition and will never receive the survival signal, as a consequence they will dye and eliminated by the macrophages. While if the affinity of the new BCR is higher than before, they will win the competition and they can choose if enter again in the cell-cycle and make another cycle, like before or differentiate in plasma cells. At this point, based on the cytokine produced by the follicular helper cells, they will undergo to isotype switch. 13 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 This B cell has to choose if differentiate in memory B cell or plasma cells, beside the one that is dying because of a lower affinity BCR. And of course, in this case there are again involved different master regulator genes and transcriptional factors. What is important to notice is that everything is driven by the CD40 signal and based on the intensity of CD40 signal BCL-6 is expressed (it is not so clear actually what is really inducing the expression of the BCL-6; BCL-6 belongs to the family of BCL2 but has nothing to do with the apoptosis. It’s a repressor transcriptional factor). If BCL-6 is expressed, it will repress the expression of Blimp-1 (that induces the differentiation in plasma cells). So, first plasma blast starts to differentiate and then produce a lot of endoplasmic reticulum and to allow the production of huge number of antibodies. This plasma blasts can remain in the lymphoid organ or migrate to the bone marrow where they fully differentiate in long live plasma cells. If BCL-6 is not present and so Blimp-1 is not expressed the B cells will become memory B cells. Probably also the absence of this other factor belonging to the IRF-4 that is necessary for the fully differentiation in plasma cells is involved in the differentiation of memory B cells. Everything is driven by CD40 and depending on the presence or absence of BCL-6: in the presence we will have memory cells while in the absence we will have the differentiation in plasma cells. Plasma cells Plasma cells are the end stage of B cells so they cannot proliferate, they are blocked in G1 and they produce a huge number of antibodies. We can also reach the 2000 immunoglobulin molecules per minutes. Of course, these cells are not only blocked in the proliferation. They cannot be anymore responsive to the T cells, so they will never do another isotype switch. So, what it is decided before in the germinal centre, they will do. They will never undergo into other cycle of hypermutations; they will produce the antibody with the high affinity, decided by the hypermutation to which is underwent the B cell during the activation and the same isotype switch. Of course, plasma cells, to be different from B cells, not express or they express a very low concentration of BCR on the cell surface, because they don’t need to recognise the antigen. Depending if they are short or long live plasma cells, they will remain few weeks in the lymphoid tissue associated to the mucosa or they will go to the bone marrow and will stay there for months. This is also based on the type of chemokine receptor expressed and decided during the differentiation, that is depending on the type of antigen and on the fact that it could be a t-dependent or t-independent activation. 14 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 This is a picture of a plasma cell, with a lot of endoplasmic reticulum but also mitochondria, Golgi and vesicles. Memory B cells monthsyear They live much more in fact they survive, like a memory T cell, for years. They can undergo randomly but infrequently on cell divisions, so sometime some self antigen can boost their activation and allow them to proliferate a bit. This is not a fully activation because we don’t need antibody against self antigen, but it is enough to maintain the constant number of memory B cells, enough to induce a faster response in the case we will encounter the same antigen to which they are specific. They have the BCR on the cell surface, in this case it is a BCR that is different, in terms of affinity, from the one that was belonging into the precursor B cells  the BCR on the membrane is different because displays the somatic mutation and a different isotype switch. All these reactions take some time, basically a couple of weeks because we need in the T-dependent antigen response, a first wave of proliferation and then the activation of the germinal centre reaction and this will take a long time. This is different to the t-independent antigen production of antibody in which after the first wave of proliferation, some B cells can differentiate in short-live plasma cells. Of course, the long live plasma cells will produce different kind of antibodies compared the first one and we have memory B cells that we don’t have before. This is the reason why now that we are very used to covid vaccine, it is known that we need to wait 10/15 days before the production of specific antibodies starts and so to be protected by the antibody’s response. This is because of the long way of the B and T cells activation. 15 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 T-independent B cell activation The T-independent B cell activation regards the B1 (that are the B cells which express on the cell surface only IgM as BCR). B1 are few and limited to a particular area: the cavity tissue and in particular the peritoneum and pleura. For the production of the antibody, they don’t require the T cell cooperation like the B2 follicular ones. They don’t go to somatic hypermutation and do never develop others antibodies, beside the senienza IgM, and of course they will never develop the immune memory. When some antigens can enter in our body, they will recognise the antigen immediately and they will produce low affinity IgM. IgM will induce a lot of effectors mechanisms important to avoid the diffusion of pathogen or even kill the pathogen. The problem in this case is that they do not develop memory and so any time we have to spend 5 days for their activation and production. These B cells will differentiate to produce the IgM in short- live plasma cells. The other B cells recognising the glycosidic antigen and so undergoing to the T- independent cell activation are the mantellar zone B2, the ones that differentiate after the Notch signal and the BAFF presence. They also express IgM on the cell surface and also a coreceptor for the BCR the CR2 that is a receptor of the complement component and which is also called CD21. This is important because it increases the activation of the BCR. The BCR is not transducing any kind of signal itself but it is associated with the coreceptor, made by Igα and Igβ small immunoglobulins, that possess inside the cytosolic tale, the classical ITAM motif (Immunoreceptor Tyrosine-based Activation Motif). So usually when the antigen is bound to the BCR and specifically many BCR are crosslinked and recruited in a small area of the cell surface, it will make the change in fosforilazione conformation of Igα and Igβ, like the TCR for the CD3. The CD45 always dephosphorylates Src kinases family (Sky) like Fyn, Lyn that will phosphorylate the ITAM motif and will allow the biding with some specific kinase that will again transduce the signal. The presence of CR2 amplifies the signal because it induces a strong change in conformation of the BCR and so it renders more faster the activation of the Syk that will also phosphorylate the ITAMs associated to the CD21. So, the CD21 is never alone, it is complexed with these two other molecules the CD19 and the CD81. The CD81 has only some transmembrane portions that are necessary to maintain all this complex bound to the membrane, but the CD19 has inside the ITAM motive. With the presence of this coreceptor we have 2 different pathways that will allow AKT and Ca2+ pathway activation to make the BCR signalling. 16 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 Here you can see that then CD19 is fosfoilated like ITAM’s motive of the coreceptor and both will lead to the activation of the B cells. Also this kind of activation takes place without the T cell cooperation and the low affinity of the BCR with the self antigen is the signal that promotes the survival of the B cells. So, the Mantellar Zone B cells are important like the B1 because the IgM produce by the MZ B cells are the one that are complexed with the antigen and complexed on the Iccosome of follicular dendritic cells. So, the first wave of production of IgM by these B cells is important to maintain and go further with the B cells activation of the follicular B cells, and so the germinal centre reaction, because they shuttled the antigen from the marginal zone to the follicular dendric cells and then triggered the high affinity immune response by the follicular B cells. As you can see here, they are in the marginal zone of the lymphoid organ where the antigen comes up and they will differentiate in plasma cells producing the IgM that then, complexed with the antigen present or the complement activated, will be (by the follicular dendritic cells trough the iccosome) showed to the B2 follicular dendritic cells that in the meanwhile already underwent to the germinal cycle centre reaction and need to compete for the signal to fully differentiate in plasma cells and memory B cells. As we said, they mainly produce IgM but, in some conditions, and specifically in the presence of pneumococcus in the lymphoid tissue associated to the mucosa, because of the strong production of some cytokines like IL-10 and TGFβ, they can produce other kind of immunoglobulins. In this case the AID transcription, that is necessary for isotype switch, is induced by the signal of BAFF that is important for their survival in the differentiation but in this case also for the AID secretion. Usually, for what concerns the humoral response, we have a steady state and constant production of natural immunoglobulins by the B1, independent from the infection. While the infection is inducing a first wave of production of antibodies by the B2 marginal zone B cells followed by a first wave of antibodies production by the follicular B2 cells, that is followed by the huge antibodies production by the fully differentiated follicular B cells. 17 Francesca Fresia/ Rachele Accastello – Lesson 13 - Immunology (Prof.ssa Paola Cappello) – 23/11/21 And in terms of isotype the natural antibodies are usually IgM. In fact, we know that B1 produce IgM. The first response of the marginal B cell responds to T -independent antigen and even the first wave of production of antibodies responds to the T-dependent antigen response. While in the stronger level of the antibodies produced after the germinal centre response, we can have IgG or different isotype. This figure shows what we’ve already said but it is also considering the second wave of infection. So we can see again the first wave we’ve just discussed. After the first time we encounter a specific antigen. The naive B celli s first activating the marginal zone, so we have IgM low affinity production level of antibody that will be after few days substituted by high affinity IgG or IgE antibody (produced by the T-dependent B follicular cells). In this case we also have the differentiation of memory B cells. You can see here like for the T cells, the memory B cells are more abundant than naive B cells, specific for the same antigen. So, when the antigen comes again these memory B cells will quickly (in 3 days instead of 7 days) induce the production of higher levels of IgG as the second wave. This is because the memory B cells will have the BCR that is an IgG and after the expansion will immediately produce plasma cells that will realise IgG. Again, memory B cells will be more abundant than in the previous wave. So, any time the response is higher, in terms of number of antibodies produced, it is faster because is related to activation of memory and not naive T cells and so the antibodies are different in isotype. It’s important to remember that the affinity of this antibody is higher in the secondary response compared to the primary response. 18 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 B CELL ACTIVATION (part 2 - end of the last lesson) This table summarizes the differences between the primary and secondary immune response: Basically, in the primary response there is a naïve B cell that encounters for the first time the antigen, so the time required for the activation is longer, because the T and B cells need to interact and decide if and how to activate this response. This time is around 1 week and about 10 days are needed to reach the peak of the response (the number of days depends on the type of the antigen). IgM are the first class of immunoglobulins that are produced and they have a lower antibody affinity. When the Memory B cells, developed from the primary immune response, encounter for the second time the same antigen, the time required to start and reach the response is shorter (about 5 days to reach the peak of the response), thanks to the phenomenon of BCR hypermutation and the higher affinity of the immune response. Thanks to the affinity maturation and the antibody class switch we have a higher immune response in comparison to the one observed in the primary immune response. Moreover, the class of the antibodies produced is also different, in fact there are IgM in the primary immune response, while in the secondary response there are IgG, IgA or IgE. Lastly, the antibody affinity is higher in the secondary response than in the primary one: this is very important to have a quick immune response, which is a typical feature of the secondary immune response. Understanding how the secondary immune response works has been very important to set up the vaccination strategy protocols: each vaccine requires different boost and different times between the first immunization and the other ones. It is fundamental to set up the protocol because the number of antibodies produced is influenced by the interval between the primary and secondary shots: - Too short, poor response → in this case, for example, our B cells are full of antigens and so they don’t have the time to do the centroblast and centrocyte cycle. - Too long, immune memory may be lost → we have to renew the memory cells For instance, the vaccine against Tetanus proved that after 25 years there’s still an immune memory, even though we generally have a booster every 10 years, just to be sure to have a good immune response. 1 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 ipersanned m In the primary immune response, to have the peak of the antibody response, the antigen dose has to reach about 106, whereas in the secondary immune response the peak is around 104 -105. This means that, thanks to hypermutation and affinity maturation of the BCR, we need very few amounts of antigen to reach the peak of the antibody response. It is also to consider that: - If the dose of the antigen is too high, we can have an exhaustion of the immune cells, as they have no time to respond well to the antigen. - If the dose of the antigen is too low, our immune cells may not recognise it and not activate a response. The route of immunization has also to be studied. For example, if the vaccine is administered orally, we will stimulate B1 and marginal zone B cells, while if we inject the vaccine intravenously, we dilute a lot the antigen and it will lose effect. The most effective route is the intramuscular one, because in this case we can use the follicular B cells, that are the ones that can collaborate with the T follicular cells and can do the hypermutation, the change of affinity and the isotype class switch. To sum up, it is important not only the time between the boosts, but also the dose of the antigen and the route of immunization. 2 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 IMMUNOGLOBULIN STRUCTURE AND ISOTYPES The first step in the study of the antibodies was made by Von Behring and Kitasato, who discovered the antitoxin effective in blocking the diphtheria disease. This discovery won the Nobel prize. Nowadays, we know a lot about antibodies, but they were firstly isolated using a normal serum that was put in an electrophoretic gel. In this way, different protein fractions were separated, starting from the albumin (the highest protein present in the serum) and the α, β and γ-globulin. If the same experiment is done with the serum of immunized patients, the γ-globulin peak increases a lot. Thanks to this test, it has been proved that antibodies are present basically into the γ-globulin peak and if we absorb the antibodies from the serum, the γ-globulin peak comes back to normal levels. Antibodies are made of 2 heavy chains and 2 light chains. Some enzymes, like Papain and Pepsin, are able to cut the antibody and are used, in some experimental procedures, to separate the part of the antibody able to bind the antigen from the constant fragment. Papain is able to cut the antibody and release the two parts able to bind the antigen separately from the constant fragment of the heavy chains. Pepsin is able to cut the antibody without separating the two light chains, but fragmenting the constant portion of the antibody, that could be degraded. It is important to know that the structure of the antibody isn’t rigid: both the arms (light chains) and the constant portion can rotate of some degrees. Thanks to this characteristic, the antibody can reach antigens that are quite distant one from another. 3 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 Here there’s the structure of the antibody with: - the V region, that is the one responsible for the binding of the antigen; it is specific for each B cells. - the C region, that confers to the immunoglobulin particular biological and functional properties; it changes during the B cells life, thanks to the isotype switching. Genetically, an individual B cell can produce only one kind of heavy chain and only one kind of light chain. Differences in the hypervariable regions of the immunoglobulin define the idiotypes of the immunoglobulin, while differences in the constant region originate different immunoglobulin isotypes and differences in constant regions of immunoglobulin due to polymorphisms can originate different immunoglobulin allotypes. Summarizing: - Differences in the hypervariable regions of an Ig define the idiotopes and the collection of idiotopes of a given Ig defines its idiotype. - Differences in constant regions of Ig molecules give rise to different Ig isotypes (2 light chain isotypes: k and λ and 5 heavy chain isotypes: μ, γ, δ, α and ε) - Differences in constant regions of Ig due to polymorphisms give rise to different Ig allotypes, thus a specific isotype can exist in different allotypic forms. 4 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 STAN L If we use an antibody as an immunogen, for example a mouse polyclonal Ig that we inject into a rabbit, we will produce rabbit anti-mouse isotypic antibodies. In blue we can see the sites recognized on the antibodies used as immunogen, that are basically present in the heavy chain and in part of the light chains. If we use as an immunogen a mouse strain A Ig and we inject it in another strain (strain B), we will produce antibodies that are mouse B anti-mouse A allotypic antibodies. The sites recognized on the antibody used as immunogen are on the heavy chain. If we use as an immunogen a strain C mouse 1 Ig injected in the same strain (strain C) into another mouse (mouse 2), we will produce mouse C anti-mouse C idiotypic antibodies. The sites are the same ones that the antibody used to recognise the antigen. There are 3 different isoforms of antibodies: 1. MEMBRANE IG → form the BCR (B cell receptor). 2. SECRETED IG → present in the blood circulation and in the lymph. arwang a 3. SECRETORY IG → present in external fluids, such as tears, mucus or saliva. MEMBRANE IMMUNOGLOBULIN: it has a transmembrane domain and an intracytoplasmic domain. SECRETED IMMUNOGLOBULIN: absence of the transmembrane and intracytoplasmic domain, there is just a tailpiece. SECRETORY IMMUNOGLOBULIN: two monomers of immunoglobulin are bound together thanks to a secretory component. 5 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 The secreted Ig can be also a dimer (as happen for the IgA) and the dimer is the result of two monomers joined together in the Fc site with the help of a J chain. This picture shows the synthesis of membrane Ig and secreted Ig. At first, there’s the synthesis of the heavy and light chains and then in the Golgi apparatus the glycosylation takes place. Membrane immunoglobulins (with their cytoplasmic and transmembrane domains) and secreted immunoglobulins emerge from the Golgi apparatus. The secreted immunoglobulins, not possessing the transmembrane and intracytoplasmic domain, can detach from the membrane. 6 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 Dimeric IgA The dimeric IgA are connected by the J chain and can bind a polymeric immunoglobulin receptor (plgR) in order to cross the epithelium and reach the mucus of the respiratory or intestinal tract. A portion of this receptor remains attached to the dimeric IgA and confers protection from the degradation that can occur in the lumen of the respiratory or intestinal tract. Ig repertoire: the binding site How can 2.0 x 104 genes code for more than 1011 BCR binding sites? A relatively small number of genes are able to produce a huge number of BCR and the generation of this immense repertoire of binding sites is the result of two main processes: 1. COMBINATORIAL DIVERSITY: different fragments can be chosen and bound together 2. JUNCTIONAL DIVERSITY: introduced by the TdT enzyme that can increase the number of BCR. What does the binding site bind? The binding site of our antibodies recognises very few sequences of the entire epitope. In fact, the antigen is the result of many epitopes that can be recognised by different antibodies. The number of epitopes expressed by an antigen is defined “antigen valence”. In the picture, there are 5 different epitopes (different colours), so the antigen valence is 5. 7 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 Moreover, the antigen epitopes can be linear (formed by a sequence of amino acids) or conformational. In nature, the antigen epitopes are most frequently conformational epitopes, made by amino acids not in sequence. If we degrade a conformational epitope to have a linear one, the antibodies that recognised the conformational one, cannot anymore recognise the same epitopes in a linear way. This is because the binding sites of the Ig cannot contact well the amino acids, as they are too distant from the arms of the Ig. Linear Conformational By sequencing the amino acids of the binding sites of the Ig, it was discovered that there are both conserved and hypervariable sequences of amino acids. In particular, in the hypervariable region are distributed in both chains, heavy and light. The chart below shows in red the hypervariable region, while in yellow and blue the conservative regions. 8 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 The hypervariable regions (in pink) are distributed in the framework region (in blue). The N-glycosylation sites are very important to confer structure to the Ig. The Hinge region allows the movements of the Ig. Since the amino acid sequences of the hypervariable region recognize a lot of antigens, they are present in the external part of the Ig (highlighted in red). This part is external because it allows the contact with different types of epitopes. Amino acid sequences of hypervariable regions form loops exposed on the external part of the Ig. These external loops are called COMPLEMENTARITY DETERMINING REGIONS (CDR) since they bind complementary regions of the antigen. As said before, the COMPLEMENTARITY DETERMINING REGIONS (CDR) of both the heavy and light chains and form the antibody binding site. 9 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 Looking more in detail the binding between the Ig (in green) and the antigen (in grey), we can see that the interaction between the antigen and the Ig is present in two points (highlighted in yellow): in this case the interaction is very low because there are just two joints. As a result of this poor interaction, the affinity (strength and persistence) of the interaction between the binding sites and the epitopes depends on their spatial (conformational) complementarity. Here we have a better complementarity between the antigen and the Ig. There are more sites of contact and the strength of this interaction results in a very high affinity and it will be more persistent. The complementarity between the Ig and the epitope depends on the 3 hypervariable regions of the heavy (in blue) and light (in red) chains. A few amino acids of heavy and light chains are involved in the direct binding with the antigen. The interaction between an immunoglobulin and an epitope is reversible and not covalent. The association between the Ig and the epitope is calculated with the “association/dissociation constant” according to the strength of the interaction. The strength by which a single binding site interacts with an epitope is called AFFINITY. Affinity of an Ig for a given monovalent antigen is evaluated by equilibrium dialysis. This test measures the amount of a monovalent antigen (in moles) required to bind 50% of the Ig binding sites. Higher is the Ig affinity for that special antigen, lower is the amount of antigen required to bind the 50% of the binding sites of the antibodies. 10 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 This is the formula that calculates the Association or Affinity Constant → higher is the antibody [Ag] affinity, it will be necessary less antigen to create the antigen-antibody complex [AgAb] THE SPACE COMPLEMENTARITY The space complementarity is an important point to consider. If the antibody and the antigen are distant, the strength of the bonds is low. On the other hand, if the antibody and the antigen are close there is a higher strength of the bond. → the strength of all these forces is inversely proportional (on log scale) to the spatial distance of the interacting atoms. The higher the distance, the lower is the strength of the binding. THE CHEMICAL BONDS The affinity between the binding site and the epitope depends on multiple, weak and non-covalent bonds. → The chemical bonds between the immunoglobulin and the binding sites are non-covalent, so basically, they can be hydrogen bonds or can be regulated by hydrophobic forces, electrostatic forces or Van De Waals forces. HYDROGEN BONDS: the hydrogen is shared between electronegative atoms. HYDROPHOBIC FORCES: the ionic groups of opposite charge pack together, excluding the water molecules. ELECTROSTATIC FORCES: the attraction between opposite charges; the strength of this interaction is regulated by Coulomb’s law. VAN DE WAALS FORCES: the fluctuation in electron clouds around the molecules polarizes the neighbouring atoms in opposite directions. The strength by which an Ig interacts with a monovalent antigen is called AFFINITY. At least, in a monomeric immunoglobulin there are 2 binding sites, while when we have for example the IgA, we have two monomers and 4 binding sites. 11 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 If the Ig has multiple binding sites, it will also have a greater binding strength and therefore a better AVIDITY, because it can bind a lot of epitopes. The AVIDITY reflets the real immunoglobulin affinity, taking into account the immunoglobulin valence, which is the ability to bind an antigen. For instance, the constant fragment of the immunoglobulin has a valence = 0, since in this portion there are not binding sites. If we consider the Fab, with one binding site, we have a valence = 1; while the entire monomeric Ig has a valence = 2. The pentameric IgM (five monomers joint together) we can reach a valence of 10 (ten binding sites). Taking into account the same affinity, an IgM can have a higher avidity in comparison to an IgG, in which there are just 2 binding sites. This is a better description of the function of the different monomers: VH and VL = variable region of the heavy and the light chains HINGE is a flexible part, thanks to the big presence of proline and cysteine. In the constant fragment of the Ig, there is a site (CH2) used by the complement to bind the Ig and this monomer is also important for the checks of the catabolism. CH3 is, instead, the site which interacts with the Fc Receptor (present on phagocytes). 12 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 Ig CLASSES Immunoglobulins are made by 5 kinds of heavy chain (α, γ, ε, δ, μ). These 5 Ig isotypes mediate different biological functions, such as the immune phagocytosis, the opsonization, the complement activation and ADCC (Antibody Dependent Cellular Cytotoxicity). Ig ACTIVITIES The activities of the Ig depend on their ability to bind their target: for example, they have the ability to neutralize a toxin, to block the viruses entering in the body or to form the immune complexes. Different other activities of the Ig are mediated by the Fc fragment that can activate the complement cascade and interact with Fc receptors expressed on the membrane of immune cells. Fc receptors can be of two different kinds: 1. Fc receptors with high affinity, able to bind the Fc portion of the Ig. 2. Fc receptors with low affinity, that bind different Ig, bound to their antigen. In this way, their interaction forms an immune complex, a structure similar to a bridge. Biological features of Ig IgG IgG are monomeric and are divided into 4 sub-classes: IgG1, IgG2, IgG3 and IgG4. Their H chain is made by four globular domains, while the Fcγ fragment is made by two globular domains. They are the most common Ig isotype present in the blood → 80% of serum Ig – 10/15 mg/ml They have a long half-life: about 20 days. The major differences among the 4 sub-classes of IgG are due to the hinge region and the intra- chain S-S bridges that confer the flexibility to the Ig. The Fcγ part of the Ig can interact with several Fcγ receptors. We can have high-affinity activating FcγR or low-affinity activating FcγR. The first ones can immobilize a monomeric IgG, while the low- affinity activating FcγR (that are 4: FcγRIIA, FcγRIIC, FcγRIIIA and FcγRIIIB) can only immobilize immune-complexed IgG. 13 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 The interaction of IgG with these FcγR triggers reactive functions of neutrophils, dendritic cells, monocytes and macrophages. One FcγR chain binds the Ig and other chains (ξ and γ) transduce the signals. IgG interaction with FcγRIII In this picture there is a low affinity activating FcγR that binds the immune complex → there is the activation of the ITAM sequence and the phosphorylation of SYK that activates the signalling transduction. As a result, there is the cell activation with the production of antibodies mediating cytotoxicity, the phagocytosis, the cytokine release or the oxidative burst. The Fcγ can also interact with another type of FcγR which is the FcγRn (neonatal γ- immunoglobulin Fc receptor) on epithelial cells of the syncytiotrophoblast. IgG interaction with FcγRn The FcγRn allows the transcytosis of maternal IgG across the placenta and their release in the fetal blood. Thanks to this mechanism, maternal IgG protect the newborn against infection for about 3 months after the delivery. Here it is represented the transfer of the IgG from the maternal blood to the fetal circulation, passing through the formation of early and acidified endosomes. 14 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 Diseases associated with maternal IgG and FcγRn As we said before, maternal IgG are important to protect the infant from infections for about 3 months after the delivery → X-linked Bruton immunodeficiency: the baby with this pathology has a block in the pro-B differentiation and therefore a deficiency in the Ig production. This disease can be discovered only after some months from the delivery, because in the first months of life the baby uses the IgG obtained from the mother. In case of alterations, such as the Bruton disease or other similar pathologies, when the maternal IgG finish the baby may develop severe pathologies. The FcγRn has another important function: in patients with Graves’ disease the mother develops antibodies against the Thyroid Stimulating Hormones (TSH) receptors, that are transmitted to the baby thanks to FcγRn. Once the baby is born, he/she has the symptoms of the Graves’ disease, but pathology can be treated by replacing the plasma with a normal one, in order to remove the maternal antibodies. Maternal IgG may also cause haemolytic disease of the newborn or the erythroblastosis fetalis → this happens when the mother is Rh– and the children is Rh+. At the first pregnancy no problems occur, but during the delivery the contact between the blood of the baby and the one of the mother sensibilizes the mother, who starts to produce antibodies against Rh+. If there is a second pregnancy, there will be a severe anaemia into the baby because the antibodies of the mother try to destroy the erythrocytes of the baby. As a result, the fetus displays high levels of bilirubin and icterus. We can prevent this pathology by administrating anti-Rh Ig after the delivery. IgG interaction with FcγRIIB The immune-complexed IgG may also bind the low affinity FcγRIIB: the signal delivered by FcγRIIB inhibits cell functions and induces cell apoptosis. The binding of IgG complexed with the antigen to the FcγRIIB on the membrane of a plasma cell delivers a death signal, inducing the apoptosis of the plasma cell and thus regulating the antibody production. The use of this inhibitory receptors is very useful to switch off the production of antibodies when they are no longer needed. This picture shows the binding of the immune complex to the receptor and the induction of apoptosis, thanks to the activation of the ITIM motif. 15 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 IgA IgA are the most abundant immunoglobulin isotype of the body. Their H chain is made by four globular domains. Monomeric IgA are the 10-15% of serum Ig. There are 2 sub-classes: IgA1 and IgA2. They are present in different ratio according to the site → IgA1: IgA2 ratio in the blood is equal to 10:1; in the small intestine 3:2 and in the colon 2:3. Monomeric IgA have a short half-life, which is about 6 days. The main difference between IgA1 and IgA2 is the O-linked carbohydrate present in the arms of the Ig that confers high mobility. This flexibility allows an efficient bivalent binding to large antigens. Apart from monomeric IgA, we can have Dimeric IgA in which we have two monomers joined in the Fc site using the J chain. The J chain is important to allow the formation of the secretory IgA. Dimeric and secretory IgA Several IgA are secreted as dimers or trimers associated to a J chain. Dimeric IgA are secreted and operate mainly on epithelial surfaces where they can interact with the polymeric Ig receptor (pIgR) that transports by transcytosis dimeric IgA to the luminal surface of the epithelia. Secretory IgA are mostly present in the gut, faeces, saliva, milk, mucus, catarrh and tears. The picture shows the structure of the polymeric Ig receptor (plgR) that is bound by the dimeric IgA. The same type of receptor is involved in the transport of IgM. 16 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 The plasma cell secretes the IgA, then there is the formation of the dimeric IgA thanks to the J chain. The dimeric IgA can bind the plgR and pass through the lumen. Only a piece of the receptor (a soluble extracellular domain), called secretory component, remains attached to the IgA and confers protection against enzymes that can be present. Pathologies related to IgA One is the IgA deficiency in which the patient has a low amount of IgA and displays a slightly higher incidence of infections (especially in the respiratory and intestinal tract, where IgA confer protection), allergies and autoimmune diseases. Basically, these patients are treated using some drugs to control the infection/allergy/autoimmune disease. In some patients, with the time, the IgA levels come back to normal levels and this leads to the disappearance of the symptoms. IgM The IgM can be monomeric → with 5 globular domains. As monomers, the IgM are expressed on the membrane of B cells where they act as BCRs. When secreted, the five IgM monomers are covalently bound by the J chain (same as IgA) creating a pentameric IgM. The pentameric IgM are 5-10% of serum Ig (0,1-1,5 mg/ml). The pentameric IgM do not cross the placenta and the binding with the poly Ig receptor (plgR) allows their transcytosis to milk and mucosal secretions. Pentameric IgM are very important because they are able to activate the complement cascade through the classical pathway. Pentameric IgM have a major role in protecting against infections in the blood stream, whereas monomeric IgM diffuse better into the tissues. IgM are the first class of antibodies produced after an infection or an immunization and they are also the first antibodies produced by the fetus. → The first Covid tests allowed the discrimination between IgM and IgG, this is because we needed to understand if the patients had an infection due to a first encounter of the covid-19 or it was a subsequent encounter. Pentameric IgM bind 5-10 epitopes and because of steric hindrance, the structure of the antigen may limit their binding capacity. Pentameric IgM are also studied for their ability to bind multiple epitopes and this make them very efficient in antigen agglutination. The presence of 5 Fcμ allows a single IgM to be sufficient to trigger the complement cascade. In fact, Fcμ interacts with the receptor FcμR (TOSO/FAIM3) present in follicular dendritic cells, macrophages and B cells. 17 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 In the picture we can see the pentameric IgM that can bind the antigen on the bacterial surface adopting the staple form. As we said before, IgM are the first natural antibodies produced by B1 cells, also in apparent absence of any infection or deliberate immunization. These natural antibodies are highly cross-reactive IgM that bind with low affinity common microbial antigens (such as phosphocholine and common constituents of bacterial cell membrane) and self-antigens. These natural IgM provide also effective means of activating the complement cascade on microbial surfaces before bacteria become dangerous. There is also a special kind of IgM: the iso-hemo-agglutinins which are able to agglutinate red blood cells (RBC) expressing A or B blood groups. A, B and 0 blood groups are made of different carbohydrate chains and these carbohydrates are expressed by RBC and body tissues. According to the presence in our body of A, B or 0 group, we will naturally develop IgM against the other blood groups. Of course, we don’t produce antibodies against the blood group that we express (self-tolerance). So, depending on the inherited genes, human RBC display distinct A, B, AB and 0 carbohydrates. Individuals belonging to the A group inherited an enzyme able to add α-N-acetyl- galactosamine to the substance H Individuals belonging to the B group inherited an enzyme able to add α-D-galactose to the substance H Individuals belonging to the AB group inherited both an enzyme able to add α-N-acetyl- galactosamine and an enzyme able to add α-D-galactose to the substance H Individuals belonging to the 0 group inherited none of these two enzymes and express only the substance H. 18 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 According to the genes that are inherited in homozygosis or heterozygosis, we can produce these enzymes that allow us to develop one of the blood groups and the different antibodies. Anti-A and anti-B antibodies are IgM and therefore they do not cross the placenta, so if the mother and the baby have different blood groups it’s not a problem. These IgM are the natural reaction against sugars of the cell wall of bacteria normally present in the gut and bronchi → this is why we develop these antibodies. The antibody response against carbohydrate antigens involves IgM only. T cells do not deliver isotype switching signals. In the following pictures, there is the distribution of the different blood groups. IgE The molecular structure is a monomer. The heavy chain is made by 5 globular domains and the Fcε by three domains. IgE are present just in traces in the serum and they also have a short half- life (2 days), but if the IgE bind the tetrameric FcεRI receptor, they can persist for a very long time (also years). The Fcε is bound by tetrameric FcεRI on the membrane of basophils and mast cells, homing just beneath the skin and mucosa and along blood vessels, and by trimeric FcεRI on the cell membrane of eosinophils and antigen presenting cells. 19 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 IgE interaction with tetrameric FcεRI The tetrameric FcεRI has an α chain that can bind with high affinity traces of IgE present in the serum. After the binding, there is the activation of ITAM sequences which are tyrosine phosphorylated after the antigen crosslinking of IgE. The simple IgE binding to FcεRI results in an increased survival of the cell The antigen-mediated crosslinking with IgE bound to FcεRI triggers a signalling cascade that leads within minutes to the release of pre-formed mediators, such as histamine, and lipid-mediator synthesis. These events produce an immediate-type allergic reaction characterized by vasodilation, increased vascular permeability, upregulation of vascular adhesion molecules and bronchoconstriction. This photograph shows an immediate and a late phase reaction → IgE interaction with trimeric FcεRI The trimeric FcεRI is expressed by eosinophils and antigen presenting cells (APC). In this case: a) The antigen-mediated crosslinking with IgE bound to FcεRI induces the degranulation of the eosinophils. b) The crosslinking of FcεRI with IgE and the antigen leads to the endocytosis of IgE-bound antigen, followed by the presentation of antigen peptides by MHC class II molecules to T cells. c) FcεRI crosslinking also induces the signalling, which leads to the production of pro-inflammatory cytokines. IgD Monomeric IgD: the heavy chain is composed by four domains. They are very flexible and they are able to form an unusual wide angle between Fab and bind distant epitopes. Secreted IgD: they are present in very low traces in serum. Membrane IgD are mostly expressed on the membrane of naive mature B cells, where IgD act as antigen receptors (BCR). 20 Rachele Accastello / Giada Fregnan – IMMUNOLOGY – Lezione 14 - Prof. Claudia Curcio – 25/11/2021 Ig classes are selectively distributed: IgG and IgM predominate in the blood. IgG and monomeric IgA are the major antibodies in the extracellular fluid within the body. Dimeric IgA prevail in secretions. The fetus receives IgG from the mother thanks to FcγRn, by transplacental transport. IgE is found mainly associated with mast cells, beneath epithelial surfaces (especially in the respiratory tract, gastrointestinal tract and skin). This table summarizes the main characteristics of each Ig subtypes: For example, the longest half-life is the one of IgG1 and IgG2, while the shorter is the one of IgD and IgE. Moreover, only IgG and IgM are able to activate the complement. 21