Immunology Lecture Notes PDF - 21/10/2021

Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 Both these molecules (HLA E and F) usually present an antigen or what is inside the cells to gamma- delta T cells that don’t recognize the classical protein peptide but glycosilate...

Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 Both these molecules (HLA E and F) usually present an antigen or what is inside the cells to gamma- delta T cells that don’t recognize the classical protein peptide but glycosilated or even glycoproteins, glycopeptides and glycolipidic antigens, which are very different from the previoous one. These molecules are less polymorphic; in fact, HLA-E molecules have only 8 alleles, the F ones only 20. The HLA-G is expresses in trofoblasts, so in the interface of mother-fetus. It is particulary important to recognize the NK cells circulating in order to protect the eventual immunoresponse against the featus. From the immunological point of view, during the pregnancy, the immunoresponse is controlled thanks to the estrogen and progesteron. The HLA-G is necessary for the inibition of the NK cells. Between the non-classical HLA I molecules there are also the MIC-A and MIC-B that we have fumorph mentioned among the stress ligands. Trasforming neoplastic cells can interact with this ligand and be recognized by the activating receptor of the NK cells. The same happens for the cells infected by the micobacteria; the micobacteria induce this expression and in this way the cells can be easly recognized by NK receptor even if they have also the express of the HLA class I molecule, but in that case it preveale the activation signal. Haemocromatsus gene (HFE) Those genes, if they are mutated, are responsible for the Haemocromatosis that is a disease in which the iron is accumulated in our cells. There is an iron receptor that bind the transferrin and also veicolate the iron complex outside the cell. This is important because we don’t need to much iron not coupled with some proteins inside the cell. We have other molecules, which are part of the non-classical ones, that are similar for the structure to the last ones but are coded by genes that are located outside the complex. These molecules are costituted by an alpha chain, a globular chain and a beta2 microglobuline. Like the non classical HLA class 1 molecule, they are less polymorphic. These molecules are involved in the immunoresponse because some of them activate NK cells like ULBP, some activate inflammation. The CD1 complex is important because it can activate the gamma delta chain, the T cells and others showing the bacterial lipid. Another receptor for the immunoglobuline is FCRN (FC receptor neonatal) that allow the traposrtation of the immunoglobuline from the mother to the fetus. C1 complex We have different molecules beloning to C1 constituted by alpha and beta2-microglobuline that show the glycolipid in their pocket, so a different kind of antigen compared to the protein shown by the classical HLA class1 molecule. The C1 express and activate, from the dendritic cells (which are the professional antigen presenting cells) and macrophages, the non classical T cells (the alpha-beta), because this is a property of only HLA class I molecules, and the NK cells. The neonatal C receptor brings inside the placenta the immunoglobuline from the mother and the ULBP protein that is expressed by the cells like MK and MB, some sort of ligands induced by the infection of cytomegalovirus (the HVA and HVB). In that way the cell which express the C receptor is recognized by NK cells. Then there is also the ZAG protein that is involved in the lipid homeostasis, this is less related with the immune response. 8 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 All these mentioned molecules are the non-classical molecules whose gene are outside the HLA class I complex. HLA CLASS II MOLECULES They are made by a heterodimer constituted by 2 chains, alfa and beta, and a transmembrane domain and a short cytoplastic domain. The two chains are little bit different; the alpha is bigger than beta. This is important when doing the western blot of these molecules, we should know that they have a different molecular weight. These class II molecules can be constitutively expressed by few cells; they are expressed only by dendritic cells, Langerhans cells epithelial cells and macrophages. Different signals, especially cytokines, can induce the expression of more transcriptional factors called class II trans activators. We will call them CIITA (class II trans activator). This transcriptional factor can be expressed and so it can induce the expression of the MHC class II molecules also in different cells (epithelial, endothelial). It can increase the expression of the macrophages or eosinophils (when we have described all the innate cells, we said that the eosinophils can be some antigen presenting cells). The fibroblasts have been demonstrated to be able to express some of these molecules and even different kind of tumour cells can express those molecules. For each molecule (DP, DQ and DR), because they are a dimer, we have two genes: alfa and beta. We have different alleles for each one. Each of us express different kind of a little molecule, the potential combination is very huge; this increases a lot the diversity of each of us (the haplotype). 9 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 The co-dominance for HLA class II molecules is different because the molecules are made of two chains. We can have 4 different results for each molecule: we have 2 different scenario that are defined cis-codominance or trans-codominance. Considering the colour code (the blue one for the father and the green and yellow for the mother), for each molecule we have 2 genes from the father that can combine together (alfa and beta), the same happens with the alpha and beta form the mother: this is what we call cis-codominance because they are forming again the same molecule present on the parent. They can also scramble and couple together: we can have the alfa chain from the father and beta chain form the mother, or the alfa chain from the mother and beta chain from the father → trans-codominance. This increases in each of us the potential combination and the expression of different molecules, even if the genes are only 6, 4 in this case and 12 in total, this 12 can be different recombined. The expression of these genes is under the control of this transcriptional factor in C28. C28 in the cells that constitutively express this molecule is induced by the presence of tissue specific stimuli, but the interferon gamma and type I can induce the expression of C28 and so the potential expression of the class II in the other type of cells. 10 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 Genetic defects on these genes lead to a serious immunodeficiency that is called the nude lymphocyte syndrome. Class II molecule is recognized by the T lymphocyte and class II is expressed on the thymic epithelial cells where the lymphocytes mature (so the class II molecule is important for the maturation of the lymphocytes). This molecule needs to be educated in recognizing what is self and what is not self. If this molecule lacks and the thymus is not expressed in this molecule, the T cells cannot recognize the CD4. Part of the cells that are recognized by this molecule are not sensible to the presence of this molecule, so they cannot mature and exit from the thymus. These people do not have a part of T lymphocyte and having not the sub population impair the complex and the general immune response; in fact, this population of T cells is necessary for the B cells activation, for the CIITA activation and these people have serious defect in the immune response. Some tissue specific factors induce the CIITA expression that can translocate into the nucleus and induce the expression of class II molecules. However, during the infection there is the production of the interferon, especially interferon gamma. The signalling from the interferon gamma receptors (JAK, STAT and so on) can induce the expression of CIITA by passing the tissue specific. In any kind of cells that have the interferon gamma receptor, the CIITA can induce the expression of MHC class II molecules. In the cells in which there are the tissue specific stimuli, the amount of the cells on the cell surface will be amplified 11 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 The peptide in the groove of HLA class II is quite longer, in this case we have 13/20 amino acids, this is due to the different form of the groove. In this image, in yellow we can see the peptide. For these molecules the groove is more open, the peptide can exit, so it’s not close like the one of the MHC class I. For that reason, the length of peptide can be different and longer. In class II molecules we don’t need the presence of anchor amino acids like before, the chemical bound is not covalent, so it’s another promiscuous binding in order to allow different peptides to be allocated in the pocket of each class II molecule. Each class II molecule present to the T cells or outside the cell surface different type of peptides. NON-CLASSICAL CLASS II MOLECULES We have other non-MHC genes inside the complex, but these are also participating in the antigen presentation process, are very related with the function of HLA molecule. The non-classical HLA class II molecules (DM and DO) are made as the classical ones, by two chains (we have DM-A, DM-B, DO-A and DO-B). These molecules don’t form the groove, so they aren’t exposes on the cell surface, but they are necessary as a chaperonin to allow the MHC class II molecules going on the cell surface. So, they are anyway participating to the antigen presentation processes, but they remain inside, and they are allowing the vehiculation of the MHC class II molecules from the different vesicles inside our cells and to the cell surface. They are important to maintain the conformation of the class II molecules until they are exposed. This non-HLA class II molecule is inside the HLA cluster (for the class I we have talked about the non-HLA molecules which are similar to the classical ones, but they are outside the complex). In this case is the opposite: they non classical molecules have no ha similar structure to HLA II molecules, but they are inside the complex gene. [The LNP2 and LNP7 are involved in the processing and transportation of the antigen to the class I molecules. They are part of the proteasome, especially the immune proteasome that cut the protein in small peptide. 12 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 The type I and type II, instead, are the transporter allowing this peptide from the cytosol to go inside the endoplasmic reticulum. The tapasine instead is inside the reticulum and allow the completion of the binding of the antigen with the class I.] → this part will be seen more in details in the next lesson. HLA CLASS III MOLECULES They are very heterogeneous because they have different functions: they are secreted molecules and they remain inside the cells. They have some polymorphism but limited compared to class I and II. Genes in the class III complex: - the one that encode for the hydroxylase, an enzyme for steroid biosynthesis (steroids are important for the modulation of the immune response) - the factors C4, C2 and B factor which belong to the complement system so they are inside the HLA complex - The lymphotoxin, a cytokine released by macrophages or contained in the granule of the neutrophil, is very important for the activation of cytotoxic T cells - TNF which is a cytokine, HLA CLASS I AND II MOLECULES: FUNCTIONS The biological identity in organ transplantation must be considered very well because, otherwise, will induce the rejection. WHAT THE POLYMORPHISM OF HLA/MHC GENES DOES? 13 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 Here there is an example of the biology of the transplant at Molinette. 14 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 We can see for example the allele expressed by a donor. For the HLA A molecule he/she has two alleles (11,24), for the B molecule the donor has two alleles in homozygosis, the 35 received from the mother and one from the father, for the HLA C 4 and 4. Some of the antigens for the class II are genotyped by the qPCR, we have the DR1, DR2 and DQ A and B and again there are two different alleles. It is important to match the potential donor with the different patient in the list to get the organ, beside the blood that should be identified just to avoid the transfusion reject for the red blood cells. 15 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 Before that they also have to try to match the HLA molecule; especially they are looking for the HLA A, B and DR; they cannot look at all the molecules because otherwise they will never never find a good recipient. There is an algorithm that is helping in this choice. The first patient that came out had at least one of the two alleles for DR similar to the one of the donor. The same happened with the HLA A and B. In contrast, the other allele is very similar for DR, but it has totally different HLA A, and only one B. This is what we mean for the high polymorphism in the population and why it has to be considered for the rejection. Polymorphism is also important because each of us can present different peptides and induce different kind of response to the same pathogen; someone respond better, with a lot of cytotoxic T cells and other ones have a weaker response because maybe they present less peptide so different epitopes that generate a different repertoire of the T cells activated. So, the polymorphism has to modulate the total reaction of the population to the microbes in general and, as a consequence, it has to control the susceptibility to the immune response. The same is true for our cell proteins, some kind of haplotypes can present to the T cells some peptides from our self-molecules that are very similar to the one from the pathogen and so they can induce an auto-reactive response. We will discuss about it because diabetes or other auto immune diseases are strictly related to the kind of haplotype of the patient. Here there is another example: The B 53 allele for the HLA class I is less represented in Europe (just 1% of people) but is very high in Gambia (25% of people present it). This because it gives an advantage to the population; in fact, it allows to better respond to the plasmodium and to protect the population against the malaria (especially juvenile). For this reason, this allele has been maintained in the population. It presents some peptides that generate the cytotoxic response against plasmodium infected cells and so this help the population living in an area in which the plasmodium is very abundant. The polymorphism of hla/mhc genes bias our selection of sexual partners: It seems that the different haplotypes have some bias for the choice of the sexual partner. This is related to some studies that have been done with different population like the hatteries in Alto Adige. There is a small population in Italy deriving from one in US like the Mormons, so a very close community in which we can imagine that the partner is closed to the community and so, for generation to generation the haplotypes are very close → it is not like that. It seems that the HLA expression is related to the expression of different olfactory receptors which are necessary for the pheromones that are very common in other species; in fact, these pheromones are caught by olfactory receptors which seems to be related and dependent to the haplotype. 16 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 In order to increase different haplotypes in population it seems that the different olfactory receptors guide the sexual choice. There is another study made in a college in which the T-shirts worn by guys for a week were presented to the girls; it seems that these girls were able to recognize the T-shirt of the proper partner thanks to the smell. This doesn’t really depend to HLA, there were not genotyped, but is only the demonstration that actually the pheromones are important in the human population. 17 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 THE ANTIGEN PROCESSING AND ANTIGEN PRESENTATION About last lesson… The two main functions of the MHC complex are: 1. To show up what is happening inside, grown or not. When the cells are infected by a bacteria or a virus, the MHC class I molecules load in their pocket the bacteria peptide. 2. The MHC molecules, class I, II and III, are the one that determine our individuality, the so- called haplotype. In this picture we can see a dendritic cell, one of the main cell type involved in the antigen presentation to the T cells. We can see how long the dendrites are. Usually the APCs (antigen presenting cells) are innate cells: macrophages, dendritic cells and B cells. Indeed, all our cells can be APCs, but we will discuss and specify when the normal (non-immune) cells can be APCs and when it is important that only an immune cell present the antigen. One of the main functions of the APCs is to take up the antigen from outside or to be directly infected by a virus or an internal bacterium, and start to show up, to the MHC class I and II molecules, the peptide related to the pathogens. This process happens inside the lymphoid organ for the naïve T cell. This means that the T cells exit from the thymus and they circulate in our body through the blood or the lymphatic circulation. A naïve or birth T cell is a T cell that has never encountered an antigen to which has a specific receptor. The APCs loaded with all the stranger peptides start to be contacted by many T cells, each with different TCR. The one with a specific TCR able to recognize the peptide and the MHC will start to contact in a stronger way the APCs, even contacting other molecules on the APC. The cytokines are important and necessary because without them the T cell cannot be really activated. To be activated, the T cell must receive the signals from the TCR and from the cytokines, released by the antigen presenting cell, at this point it will proliferate and expand to reach the infected or damaged area and kill the eventual pathogen. There are two important steps for the activation (this part will be better discussed in another lesson) 1. The antigen presentation to naïve T cells in lymphoid organs 2. The effector T cell, generated after the expansion of the single T cell, will reach the tissue in the periphery and there the epithelial and mucosa infected cells will show the viral peptide to the MHC class I and II. At this point, these cells will be recognized and killed by the effector T cell. 1 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 THE ANTIGEN PRESENTATION The best antigen presenting cells are the dendritic cells, they are called professional APCs. These cells are located everywhere (ex. in the skin, in the mucosa, in the respiratory and intestinal tract, etc.). Features: - They can easily and quickly catch up the microbes. - They have the receptors PRR (the complement receptor) so they can easily phagocytise through the normal PRR, but they can also phagocytise everything that is opsonised by the complement. - They are able to migrate, so they are not stacked in the tissues. - After the migration they start a program of maturation, in fact in the tissues they are immature cells. Once mature, they transform themselves in antigen presenting cells. They have to migrate where the T cells are more abundant in the lymphoid organ. - To perform the maturation, they start to express a very important chemokine receptor (CCR7) which is in common with the naïve T cells’ receptor used to enter in the lymphoid tissues. - Only after the expression of the CCR7 they can start a peculiar maturation program through which they change their status from immature to mature. Because they are the professional APCs, the dendritic cells have also a practical application in clinic: many tumour protocols use the dendritic cells. We can take the peripheral blood from a patient with a tumour, in that way we will have both the tumour and the dendritic cells. These cells then migrate into the lymph node where they encounter a lot of naïve T cells, even memory cells, to which they present the antigen, in that way they can reactivate the new T cells. The only phagocytosis occurs during the maturation process: dendritic cells start to mature and express chemokine receptors (CCR7) which are important to the migration to the lymphoid organs; CCL19 and CCL21 are constitutively expressed and needed to recruit naive T cells and dendritic cells. During the travelling through the lymphatic circulation, they change the expression of some membrane molecules, such as a major expression of class I or class II molecules or major expression of co-stimulatory molecules. The dendritic cells are usually contacted by several T cells, but only those with the compatible TCR will interact. The dendritic cells can capture the antigen through the PRR; in fact, when they are immature they are specialized in phagocytosis → this process starts their conversion in mature dendritic cells. 2 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 The conversion in mature dendritic cells is conducted by the expression of peptides in association with MHC class I or II, alarm signals, the modulation of the repertoire of cytokines expressed by the APC and so on; however, we have not only the expansion of T cells, but also the differentiation of those cells thanks to different cytokines. In this way there is the formation of a starting APC specialized in the capture the antigen. This scheme summarizes the process. 3 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 DIFFERENCES BETWEEN THE APCs Not only the dendritic cells can be APC, but there are also the macrophages and the B cells. In this table it is possible to appreciate the differences between the APCs. 1. The antigen uptake is quite good in every kind of cells. 2. The B cells are not able to phagocytose, in those cells, it is the BCR (the immunoglobulin on the membrane) that specifically binds the antigen leading the B cells to the endocytosis. This way, B cells can activate themselves. For dendritic cells and macrophages this process is focused on the activation of T cells, on B cells instead the presentation of the antigen helps themselves in the production of a lot of antibodies. 3. Considering the level of expression of the MHC complex, it is different between these cells. All of them can constitutively express MHC class II but the macrophages and the B cells start to express this molecule after having received some signals, so they are inducible. 4. The dendritic cells, after the maturation, will present all the constitutive molecules for the co-stimulation delivery, which are only inducible on macrophages and B cells 5. The location is different, in fact the dendritic cells are everywhere, the macrophages are found in different tissues (the lymphoid tissue, connective tissue, the pleura, the pericardium, etc.) and the B cells are only present in the lymphoid tissue. The dendritic cells are the only one able to activate the naïve T cells, while macrophages and B cells can activate only the T cells which are pre-activated by the dendritic cells, NOT naive T cells. What is showed up from the MHC class I molecules? Usually, short peptides which derive from an endogenous protein (our proteins, endogenous bacteria, viruses or some tumoral antigens) are presented. CD8T cells, which have a killing function, recognize intracellular peptides and this recognition leads to the direct target cell disruption. As for peptides derived from exogenous proteins (generated by the phagocytosis), they are processed and presented in the pocket of MHC class II molecules. These are then recognized by CD4T cells, which have a helper function; in fact, they can secrete cytokines which are important to communicate with all the immune cells and to upregulate all the MHC class I or II molecules or the co-stimulating molecules. The cytokines are also important in the lymphoid organs to generate the killing cells (CD8T cells) and are important to allow the B cells to secrete a specific type of antibody. 4 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 PRESENTATION OF ENDOGENUS PEPTIDES A single cell contains several proteins which can be easily targeted by ubiquitin ligases and easily recognized and degraded by the proteasome. The ubiquitin modifies the tertiary structure of the protein to enter this channel, the proteasome. The proteasome, as we can see in the image, is a channel made of six subunits with protease activity: only proteins characterized by a wrong structure (e.g., incorrect folding) can be recognized by the ubiquitin and be degraded in small peptides, which are NOT ready to be loaded on APCs. The structure of the proteasome can change during the infection, as the interferon gamma (usually produced during viral or endogenous bacterial infection) is able to increase the expression of two different components of the proteasome: LMP2 and LMP7. LMP2 and LMP7 are slightly polymorphic whose genes are located inside the HLA class II complex; they can change the proteasome in an immunoproteasome which is able to cut the hydrophobic and residues necessary to the binding of these peptides on APCs. The immunoproteasome is more prone to produce the peptides allocated in the MHC class I groove. The main difference between these two (as we can see in the upper image) is represented by the fact that proteasomes are able to cut only proteins targeted by the ubiquitin, generating some peptides but also amino acids and residues that are usually bind?? by our cells, while the immunoproteasomes, in the presence of cytokines, change the conformation and become able to cut those proteins which are not ubiquitinated in order to produce small peptides for the binding to the APCs (only in presence of specific signals!). The function of the immunoproteasome is important because during the viral replication we don’t waste time by activating the ubiquitin ligase. 5 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 These peptides coming out from the proteasome are quickly digested in the cytosol by peptidase or aminopeptidase, otherwise they can be dangerous for the cell; however, some peptides manage to rescue themselves thanks to the presence of specific transmembrane transporters located on the ER membrane. These transporters allow the entrance of these peptides in the reticulum. These transporters are made by two molecules: TAP1 and TAP2, whose genes are located in the MHC class II complex (increase variability of transports in the population). MHC class I is synthesized also in the reticulum, and it is made only by the alpha chain that needs to be bound to β2-microglobulin to maintain the right 3D structure. At this point, the pocket opens for the allocation of the peptide: when the class I molecules are synthesized, they are initially associated to a chaperonin (to maintain the 3D structure) called calnexin and calreticulin until the synthesis of β2-microglobulin; this molecule substitutes the chaperonins. At this point, another chaperonin called tapasine supports MHC class I molecules. The binding with the tapasine allows all the peptides to enter the reticulum thanks the TAP transporters. 6 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 All the molecules, MHC class I, β2 and the peptides, are presented to the cellular membrane through normal vesicles transport, which implies Golgi for post-translational modifications. PRESENTATION OF EXOGENOUS PEPTIDES (CLASS II) MHC class II molecules show peptides generated by exogenous proteins which derive from outside. In the image, we can see the invagination of the membrane and the formation of the phagosome that will fuse with the lysosome; in these vesicles there is the degradation of the proteins without the involvement of the proteasome. In the meanwhile, there is the synthesis of the MHC class II molecules inside some vesicles that have to fuse with the ones containing the peptides; this process allows the binding of all these peptides, generated by the enzymatic degradation, to the pocket of the MHC class II molecule. Then, through the process of exocytosis they can be exposed on the cell surface. 7 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 Alpha and beta chains of MHC class II molecules are encoded by different genes and independently synthetized in the reticulum where they are maintained in their right 3D structure as they have pockets which need to bind something that is exogenous and not derived from an already existent gene. To maintain the right structure the presence of the invariant chain is very important, which fills and protects the pocket of the MHC class II complex; however, it has also another function that is the construction of three HLA class II molecules (DP, DM, DO) through its trimerization domain; in fact, the invariant chain has a small peptide called clip which occupies the groove, and the trimerization domain. These 3 molecules are transported from the ER to the Golgi and post-Golgi vesicles without binding anything in their travel; this is important to avoid the binding with the endogenous peptides generated by the proteasome and the entrance in the ER. If the 3 molecules start to bind the peptides, they will have the groove full and they will not be able to bind the ones coming from the degraded phagocytose proteins, so they won’t show up what comes from the outside, as in the case of extracellular bacteria or fungi. Then, through the involvement of other proteins, such as the chaperonin, dynamin, and kinesin, the 3 molecules start to be transported to the post-Golgi vesicles (also called MHC class II post- Golgi vesicles) which associate to microtubules and move to the membrane surface. The presence of cathepsin will start to degrade the invariant chain, leaving only short peptides called clip inside the groove. The right conformation of the MHC class II molecules is maintained by the DM or DO molecules. After that, the vesicles are fused with the lysosome or phagosome in which the peptide become more abundant, it can bind the grove of the MHC class II molecules, the DM molecules will be detached, and the peptide will be showed on the cell surface. 8 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 The following picture summarizes all these steps: Actually, there are 4 major pathways of antigen processing. Other than the exogenous and the endogenous pathways, there are two pathways called autophagy (degradation of the organelles) and cross-presentation. 9 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 AUTHOPHAGY: it allows endogenous proteins to be loaded on the MHC class II complex and presented to the CD4 T cells which will help the B cells to produce antibodies against these proteins. CROSS-PRESENTATION: it is performed only by dendritic cells; it allows the presentation of proteins and peptides coming from the external environment (after the phagocytosis of a pathogen) to the groove of MHC class I molecules and so to the CD8 T cells (killer T cells). Dendritic cells are able to express Sec61 which is a protein that form a pore channel in the phagosomes and allows some of the peptides present inside the phagolysosome to reach the cytosol; as usual, these peptides can be then degraded by the proteasome, transported by the TAP1 and TAP2 in the reticulum and be presented in the groove of MHC class I molecules. To summarize: Exogenous protein Phagosome fuses with lysosomes Sec61, present on dendritic cells and on the membrane of endocytic vesicles, allows the exit of some of those proteins which can be showed to the MHC class I molecules and to the CD8T cells instead of the CD4T cells. Thanks to this, dangerous exogenous proteins can be directly killed. Due to the specific conformation of the groove, the MHC class I and II molecules bind different peptides; in particular, the MHC class II bind short peptides, while longer ones are bound by the class II molecules. Among all peptides, which one associate to HLA class I and class II molecules? All HLA-B27 can be bound only if they have specific amino acids (arginine and alanine) on specific positions. This is called the MHC AFFINITY → The binding of peptides to the HLA groove involves many weak binding forces. While the TCR binding to the complex (peptide and HLA) is called AVIDITY, and each specific TCR has a great avidity only for one type of complex, while HLA can bind thousands of different peptides. Furthermore, the TCR is not only binding the peptide, but also the HLA molecule: this explains what happens after the transplantation of an organ with a different MHC in a patient. The T cells of the patient recognize as a stranger molecule the HLA, so the organism can reject the organ. For the same reason, during the maturation of the T cells in the thymus, all the T cells, that bind and recognize HLA class I and class II molecules, can be rescued from elimination. Each TCR presenting on one lymphocyte can recognize the same MHC and the same peptide, but the same MHC can bind thousands of different peptides --> MHC RESTRICTION. 10 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 This concept was demonstrated by Zinkernagel and Doherty: 1. They took a strain A mouse. These mice show the same MHC molecules. 2. They infected this strain mouse with the LCMV (lymphocytic choriomeningitis virus) 3. They stimulated the anti-viral response in this mouse 4. After some weeks they took the splenocytes (the spleen is the bigger lymphoid organ from which is possible to take the T cells) among which some are specific for the infection. 5. They performed a cytotoxic assay, with these splenocytes, using two different targets: dendritic cells coming from mice of the strain A and dendritic cells coming from mice of a different strain (B). Both strains were infected in order to present the same viral antigen. What happened after this assay? - By using specific lymphocytes with the autologous dendritic cells showing the viral antigen → they saw a perfect lysis of target cells - By using the same APC, but generated by a non-infected mouse of the same strain → no lysis because the CTLs don't have recognized the self-peptide and the MHC self - By using APC coming from strain B infected with the same virus → antigen is the same, but MHC molecules are different: no lysis This demonstrates that TCRs are specific for both MHC and antigens. 11 Michelle Guichardaz / Daniele Friolotto – Lesson 7 – Immunology (Prof.ssa Paola Cappello) – 26/10/2021 THE LIPID ANTIGEN PRESENTATION Apart from proteins, also other kind of molecules can present different antigens, such as the lipidic one. This happen through different molecules (CD1) which are considered non-classical HLA molecules (whose genes are inside the class I complex) are made by an alpha chain that binds the β2-microglobulin. The groove is bound by the lipid residues coming from the digestion inside the phagolysosome; so, during the endocytosis process, the groove is loaded with the antigens; it is not important which endosome they encounter, they will show up the lipidic antigen. This complex is not specifically recognized by the alpha and beta CD8T cells, but by gamma-delta T cells which are a small population of T cells. The 85% of our T cells express a receptor made by alpha and beta chains. 50% of our lymphocytes express a TCR receptor made by two different chains: gamma and delta. The main difference is that the alpha and beta T cells can recognize the proteins through the MHC class I and II, while the gamma-delta and iNKT cells (invariant NKT cells) can recognize lipidic antigens through the binding of CD1 molecules; iNKT are similar to NK cells (they kill the cytokines and share many activating immunoreceptors of the NK cell), but like T cells they can express an invariant TCR. iNKT are more abundant in tissues than in the blood or the lymphoid organs and interact specifically with lipidic antigen and phospho-proteins. 12