Immunology Lesson 4 PDF

Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 The ligand then activates the TRADD or FADD domain: these are dead domains (characteristic of the FAS and TNF receptor family) and usually the molecules bind these adaptors in the cytosolic tail of...

Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 The ligand then activates the TRADD or FADD domain: these are dead domains (characteristic of the FAS and TNF receptor family) and usually the molecules bind these adaptors in the cytosolic tail of the receptor, and activate the caspase. To sum everything up:  The only homodimer receptors are the G-CSF;  β common chain: the β is the same for every receptor, the α chain is the specific one;  γ common chain: the γ chain is identical for all receptors, the α and the β chains change. This receptor is common for a large variety of IL, allowing different functions;  LIF (leukemia inhibitor factors) and IL-6 receptors: they are a heterodimer where the specific chain recognizes and binds the cytokine, while the transduction chain is called gp130. When the γ-chain is mutated and sometimes lacking, a lot of several complications can be observed leading to a sever combined immunodeficiency (too many cytokines are involved  many effectors don’t work). 8 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 After the binding with the ligand (cytokine), the JAKs transphosphorylate each other and, thanks to a conformational change of the receptor, they activate, by phosphorylation, the internal tail of the receptor that become the attract point for the STAT. There are other receptors that can activate some specific tyrosine kinase and induce the calcium release (and the MAPK pathway). Calcium is important because it mediates the de-granulation of granulocytes, allows the cytoskeletal actin re-organization and can migrate into the nucleus to start the transcription of new genes. Receptors can also induce the SOCS (suppressor of cytokine signaling family) by directly inhibiting the JAK activation (by interacting with JAK they don’t allow the interaction with STAT) or by inducing the ubiquitination and proteosome degradation of STAT.  the expression of that cytokine is stopped. THE IL2 RECEPTORS: They belong to the γ common chain receptor family; when the cell is not active, this receptor is made only by two chains (the β and γ); When it is switched on, it expresses a third chain, the α one, that is also called CD25 (cluster of differentiation-25). The expression of the α chain dramatically increases the receptor affinity for IL2 compared to the β one. In resting lymphocytes, a much higher number of IL2 is needed to bind the receptor (it has a lower affinity): only when IL2 is secreted in a consistent concentration, the ligand can bind the β and then, the γ chain, starting the activation of JAK/STAT. On the other hand, when lymphocytes are pre-activated through the TCR receptors, they can start producing the α chain (a mandatory step for activation during TCR recognition), which has a higher affinity for the IL2. This strategy allows to get an andvantage for some lymphocites among others in the lymphoid tissue: having a high-affinity receptor for IL2, this means they need less amount of IL2 to be activated, therefore winning the competition inside the lymphoid organ. To allow the specific action of IL2, cells can:  Express the α chain; it can also be released after a while and therefore bind the IL2 free in the cytosol. By doing this, it subtracts those cytokines to the other lymphocytes.  Use a polarized secretion of IL2 (that will start the JAK/STAT pathway which is always associated to the β and γ chains). 9 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 Some of IL belonging to the γ common chain family. It is now quite understandable why the lack of interferon γ common chain cause a severe immune deficiency in people where these genes are mutated. This immage explain the pleiotropic effect of the cytokines: they affect different kinds of cells and each of them activate in a specific way a certain type of differentiation or cells activation. Cytokines are active mainly on immune cells but can effect also fibroblast, endothelial cells and even the neuronal ones. 10 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 A very useful scheme that summarizes all the function of the cytokines: Some videos regarding:  Cytokines https://www.youtube.com/watch?v=7g2f0dhp2w8  Cytokine receptors: https://www.youtube.com/watch?v=Ms_ehUVvKKk // https://www.youtube.com/watch?v=FTbWJ0xdIuw CHEMOKINES They are a huge family (about 40 small cytokines) characterized by the ability to mediate the chemotaxis which is the direct movement of the cells responding to a chemical stimulus. This feature is common to all organism, from the simple ones, like bacteria, to more complex ones. Chemokines generate the chemical gradient that drives the recruitment of immune cells from the blood to the site of infection. The 40 members of chemokines are divided in 4 families:  Alpha family;  Beta family;  Two other families with only one member. 11 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 These first two families are now called respectively CXCL and CCL families; the re-naming is due to their different structure: in the CC, the cysteine involved in the di-sulfide bond are near one to each other; in the CXC some space is present between the two cysteines and it is occupied by an aminoacid. Some chemokines are constitutively expressed in certain tissues, especially in the lymphoid ones, in which CCL19, CCL21, CXL12 and CXCL13, attract T and B cells as well as the dendritic ones. The first two (CCL19 and CCL21) lead T lymphocytes to concentrate in a certain area of the lymphoid organs (separating the B area from the T area). The CXL12/13, on the other hand, drive the movement of the B cells that are concentrate in another zone of the lymphoid organ. Chemokines’ receptors are specifically expressed in these kinds of cells. Besides constitutively chemokines, there are also present the inflammatory ones which are produced in response to a certain stimuli (damage of the endothelial cells or the recognition of the PRRs); their function is to recruit the immune cells at the inflammatory site. Chemokines share the same features of the cytokines and so:  They are pleiotropic;  They can perform different functions in different cells: CCL5, for example, in the eosinophils will induce the degranulation but in NK cells, it drives the movement to the inflammatory site. They bind a 7-transmembrane receptor associated with a G-protein; after the binding with the chemokines, GDP is phosphorylated in GTP and so disactivated. 12 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 Two chemokines’ receptors, CXCR4 and CCR5, are also recognized by the HIV: after the HIV infection, people display a rapid decrease in CD4 cells. These are the first to contact the gp120 protein present on the viral capsid and allow the adhesion with the lymphocytes. After that, other capsid proteins bind the chemokines’ receptors (CD4 stops the virus on the cell’s surface) and therefore the infection can occur. It has been demonstrated that people with a mutation in CCR5 has a lower possibility to be infected by HIV. The frequency of this mutation is not so high, however it is more spread in the northern Europe comparing to Africa. 13 Larisa Laios / Francesco Grossi - Lesson 5 – Immunology - Prof. Capello - 19/10/21 THE COMPLEMENT SYSTEM The complement system was first identified in the early 1900’s by Jules Bordet, by studying the animal response to different pathogens. In his experiments, he put the mice serum in contact with different bacteria and observed that the serum was able to induce its killing. Once the serum was left accidentaly in the Sun (therefore heated at about 56 degrees), and then used again in his experiments, Bordet noticed that the serum was no longer effective, probably due to it being too old (he thought). Next, he mixed this “old serum” with a sample of fresh one: lysis of bacteria was observed again. His conclusion was that there was probably something from the old serum that complemented the humoral activity of the fresh one. Remember that the two main theories of that time were: the first, which suggested that the killing of the bacteria was due to a humoral response, while the second indicated a cell-mediated response. This “something” were complement proteins, which denaturate when heated. We know now that the Complement system is a collection of more that 30 proteins (proteases) present in bodily fluids, which activate the cleavage cascade by three different pathways:  Classical Pathway: triggered by the conformational change og IgM and IgG. This was the first one to be discovered.  Lectinic Pathway.  Alternative Pathway. The last two are usually triggered by microbial sugar or proteins present on viral particles. When the complement proteins are activated, they cleave and activate the next inactive protase. The process of activation (cascade sequence) only lasts a short period of time (in healthy cells) and ends with the activation af all the proteins of the system. This cascade is limited by Complement Control Proteins that keep the activation local. Each component, during the cleavage, is cut in two parts: one is linked to the cell surface (whether it’s the bacteria itself or the infected cell) and it’s the true active protease, while the smaller one is released and triggers a local inflammation. The components are identified by the letter “C” plus a number (C1, C3, C4); the number only indicates the order in which they had been discovered, it does not reflect the order of activation. The main goal of this activation is to form a pore through the pathogen, which will eventually induce osmolarity lysis. As previously mentioned, this can be achieved by different pathways. THE CLASSICAL PATHWAY The protagonist is the C1 component, especially C1q. Larisa Laios / Francesco Grossi - Lesson 5 – Immunology - Prof. Capello - 19/10/21 C1q has a particular structure: it’s made up by 6 glomerular heads, two of which need to bind an antibody each (two antibodies in total). This condition has to be met for two main reasons: if an aspecific binding occurs, the activation becomes impossible because the antibody quickly detaches from the head; secondly, if there are not enough antibodies to allow both heads to bind, it means that there is no relevant pathogen to react to. C1q itself does not have a proteolytic effect; its only purpose is to bind the antibodies and then activate the true proteolytic enzymes (C1r and C1s) by the acquisition of a serin-esterase activity. Then, the serine protease activity acquired by the activated C1q,r,s acts on the two subsequent components of the complement cascade: C4 and C2 get cut in two fragments. C4, when cut, results in a bigger fragment, C4b, which exposes a reactive group that binds covalently the membrane of the cell or the pathogen’s. The smaller fragment remains soluble. Similarly, the C2 component gets cleaved and results in two fragments: C2a (the bigger one, which then associates with C1b) and a smaller soluble portion, C2b. The combined activity of C4b and C2a (C4b2a) acquires a very important enzymatic activity called C3 convertase. This newly formed enzyme cuts C3 in two fragments (C3a and C3b as the biggest fragment). A new complex is then formed: C3b connects to C4b2a, while C3a remains soluble. The novel C4a2b3a is the C5 convertase: this goes on to cleave the C5 in C5a and C5b. Larisa Laios / Francesco Grossi - Lesson 5 – Immunology - Prof. Capello - 19/10/21 The most abundant enzyme of the complement system is C3, present in blood (1.2 mg/ml) and other bodily fluids (tears, saliva and interstitial fluids). A single C3 convertase generates more than 1000 C3b molecules, and by his action on C5 and the next generation of C5b, C5b initiates the assembly of the MAC (Membrane Attack Complex), the pore poking the pathogen. The table shown here illustrates the different human immunoglobulins. 5 different classes of Ig’s (IgG, IgM, IgA, IgD, IgE) are organized in more sub-classes. Each sub-class can activate the complement in different potencies: the classic pathway is very-well activated by the IgM and IgG3. This aspect is crucial when developing monoclonal antibodies against a disease, since ag IgM isophorm would be the most efficient compared to the other sub-types. THE LECTINIC PATHWAY This pathway is activated by various sensor proteins soluble in our bodily fluids, which we already encountered when discussing the anatomic barriers. These are the Mannose Binding Protein (MBP) and Ficolins, which recognize foreign sugars on microbe surfaces. Similarly to C1q, these proteins tend to aggregate and share a tridimentional structure.  MBP: This is a large multimeric (3 monomers) protein, where the basic monomer is made by a collagen-like tail linked to a C-lectin head acting as the carbohydrate- recognition domain.  Ficolins: these are a large family of proteins more abundant than MBP. Their basic structure is similar to MBP, however, their carbohydrate-recognizing domain is made a fibrinogen-like head. Thus, microbial sugars recognized by Ficolins are different from those recognized by MBP. When MBP and Ficolins interact with microbial sugars, they change conformation and activate the MASP (Mannan-Binding Lectin Associated Serin Protease) that adheres to the membrane, cleaves C4 into C4a and C4b and C2 into C2a and C2b. As we previously seen in the classical pathway, these components interact and form the C4b2a complex, also known as the C3 convertase of the lectinic pathway. The pathway then goes on as usual with the cleavage of C3 and C5. minuto v6 Larisa Laios / Francesco Grossi - Lesson 5 – Immunology - Prof. Capello - 19/10/21 THE ALTERNATIVE PATHWAY It’s the philologically most ancient pathway of the three and it’s triggered by the ability of the C3 to hydrolyze itself in liquid phase by exposing his thioester domain to the other components of the pathway, in order to get cleaved in C3a and C3b. If there is no infection and therefore no microbial surface to bind to, the C3b component is unable to remain stabilized and it dismutes in an inactive C3 component. When an infection is present, C3b is stabilized and binds Factor B, another component of the complement system. Factor B now bound to the fragment of C3b is then cleaved by Factor D into fragments Ba (which gests eventually released) and Bb. The Bb fragment binds the C3b component, and the Bb- C3b complex acquires a short-lived C3 convertase activity. Quick note: as we are discussing an “alternative” pathway, we are also using alternative names for different component elements with the same mechanism (Factor D is to be compared to C2). The very short life of C3bBb convertase is prolonged by another Complement factor, Properdin, which is made by neutrophils and stored in secondary granules. Properdin allows the complex to form the C5 convertase of the alternative pathway. A summary: After the cleavage of C5 in C5a and C5b by the C5 convertase, C5b recruits C6 and C7 to the membrane. But at this point, they are just bound to the cell surface, not poking it in any way. The arrival of the C8 component is needed: C8 invades the bi-layer of microbial sugar while C9 finally forms a hole trough it entirely crossing the surface. The hole interrupts the functional structure of the membrane, leading to the osmotic lysis that destroys the pathogen. Larisa Laios / Francesco Grossi - Lesson 5 – Immunology - Prof. Capello - 19/10/21 Functional meaning of the Complement System: SS  Gminues Cell killing: especially towards the Neisseria phylum, which did not evolve a thick sugary wall as other bacteria did.  Opsonization: bacteria that are able to resist the formation of the pore through the wall get coated with complement fragments or antibodies that ameliorate phagocytosis at least 10- 100 times more.  Induction (or amplification) of a local inflammatory response: this is possible because of the soluble complement fragments that get released (all the C#a) and are not involved in the generation of a convertase and the assembly of the MAC. These fragments are referred to as anaphylatoxins. They induce a very potent and hyper-sensitive reaction (similar to anaphylaxis) on receptors expressed by granulocytes, macrophages, dentritic cells and endothelial cells. They are also able to increase the size and permeability of local blood vessels (by the release of histamine and serotonine) and elicit the recruitment of inflammatory cells along with the oxidative burst in phagocytes.  Immunocomplex removal: immunocomplexes are the complexes formed by antibodies and antigens, which are able to clot and deposit in glomerulocites and thin blood vessels. These µ complexes need to be removed before an unecessary activation of the Complement that can lead to auto-destruction. This removal process is allowed by the binding of the C3b to the antibody, which can be recognized by the C1r (C1 receptor) on the red blood cell and then carried towards the spleen or the liver (where the phagocites usually degrade the red blood cells along with the complex). This table highligts the different receptors and the ligands they can bind to. Interestingly, C3b can be bound by a receptor (CD21) which acts as a co-receptor for B-Cells. This causes an amplification of the signal for the activation of the B-Cells. Some receptors can act as adhesion molecules and promote leucocite adhesion to endothelial cells. REGULATION OF THE COMPLEMENT The Complement System can be finely regulated in three different ways by the RCA (Regulator of Complement Activation):  C1 inhibitor (C1 INH): It’s a soluble molecule that inhibits C1 activation. It’s a serpin that binds C1r and C1s mimicking C4. When this regulator is absent due to a genetic inherited condition or other deficiency, this condition causes an increase in the concentration of C2a in the serum causing the hereditary angioedema, in which endothelial cells are more sensitive and tend to lysis more frequently during the complement activation.  MCP (CD64), CR1, DAF (Decay Accelerating Factor), C4b binding protein, H factor and I factor: these proteins limit the C3 and C5 convertase activity by competing with its  Larisa Laios / Francesco Grossi - Lesson 5 – Immunology - Prof. Capello - 19/10/21 substrates or by dissociating activated b complexes. The DAF factor is expressed on cell surfaces bound to a GPI; deficiency in GPI expression in HSC leads to the absence of many surface molecules and the paroxysmal nocturnal hemoglobinuria : red blood cells get destroyed more frequently during the night when temperatures are lower.  CD59, S Protein: CD59 is a surface protein physiologically present on our cells, while the S protein is a soluble factor. They inhibit the last stage of the formation of the MAP, in particular the formation of the complex made up by C8 and C9, therefore the assembly of the pore structure. The S protein has a similar activity, but it binds the C6 and C7, preventing them from inserting themselves into the membrane. COMPLEMENT DEFICIENCY  The most frequent one is related to C2.  Deficiency related to C1, C3 and C4 leads to an autoimmune disease to the impossibility to remove the immunocomplexes, causing symptoms comparable to Lupus.  Deficiency in C3b is the most serious one: not only the formation of the pore is impossible, but also no phagocytosis mediated by the complement, no removal of immunocomplexes.  Deficiency in properdin, MBL, MASP increase the risk of Meningococcical infections.  A similar problem can be induced by the absence of the regulators mentioned before (factor I, H) all related to the impossibility to remove the immunocomplexes.  Deficiency in receptors: the endothelial adhesion of leukocytes is compromised due to an absence of two surface receptors (CR3 and CR4). This leads to a less-effective or malfunctioning recruitment of leucocytes to the infection site 2,11 41.21A Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 COMMUNICATION CODES molecules 3class ofthis esortissens giàPIO Today we will discuss a new topic which we have already introduced when we have talked about the communication between immune cell and the epithelial or endocrine cells; we have a lot of e communication codes represented by the alarmin (the alarming signal), which are perceived by the PRR (Pattern recognition receptor) and are mostly present on the innate immune cell. These codes can perceive this alarm also send by all the tissue cells or our cells that are damaged or infected. We also have hormones; sexual hormones have a strong impact in immune system; men and women are very different in immune response in terms of intensity and strength for many reasons: - some 2 and 9 toll like receptor genes, are in the X chromosome - until the menopause women have a stronger immune response because of the oestrogen and so, since the level of oestrogen change, they have different intense response; woman have more circulating B than man and they have more CD4 and CD8. Cytokines They are usually secreted by the immune cell to communicate with other immune cells, they can also be secreted and released by epithelial cells. The response cells are the one that specifically express the receptor, that can also be induced by the inflammation, by the presence of other cytokines, or it can be constitutive. MHC ANTIGEN (OR HLA ANTIGEN) Oregustin immune sunon immune Some kind of HLA antigens are present in non-nucleidic cells or in specific immune cells. They are necessary to communicate with the T cells, so they are the real bridge between innate and MMMM adaptive immune response. The next time we will discuss the process of the antigen presentation, so we will discuss also the other membran ligands that are strictly related to the same process. THE MHC COMPLEX (OR MAJOR HISTOCOMPATIBILY COMPLEX) Microbes invade our organism because of the good envrioment. Before that thay have a very stressful trip and so they can be killed even before the entrance. I We have a lot of molecules, such as the defensine, and the biochemical and anatomical barriers that can directly kill the microbe. They can also be eaten, after opsonitation for example, by fagocytes; so microbes have to figure out how to enter and protect themselves from all these mechanisms. 1  Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 They have to cross the barrier and living site; in particular, they to cross the plasma membrane and live inside the cell. This is the most protected envrioment, so they have to figure out different strategies; it is a sort of evolution to enter inside and be hide by the immune system. 1 come capire cosa succede nelle nostre cellule we need ho The problem for the organism was how to catch all the microbes inside our cells. surfershow whatis Maggy theyareanagenprotein thateachoredisplaysufferer setofthisarent Ahoy The human antigen is the HLA and is ha the sam meaning of MHC. The our difference is that MHC is for all the speacies, while the HLA is only for humans (= human leukocyte antigen) Come capire cosa succede DENTRO nascosto nelle nostrecellule 2 si è evn questa ragione II.int glycoproteins showwhat happened quando sono infette danneggiate Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 singlemaestro 3 important features ei tratto o moiewn i - The MHC molecules are glicoproteins, beloning to Ig immunoglobulin family, and they are transmembran proteins express on cell surface. There are three classes of MHC molecules: I, II and III. For the critical function of showing what happening inside the cells are very important the glicoproteins call class I and class II. The first 2 are important for the presentation of what is inside, while the third are involved in immune response but not as presenter of what is inside. We have alreay said that they are present in all vertebrates, the locus of these genes are actually a cluter of genes. There is another class of these molecules, the so called non-classical MHC. They are very similar from a genetic point of view. The difference is that the non-classical do not participate in the antigen presentation. The cluster of genes in the complex is constituted of a lot of loci, so it is polygenic. However, it is also polymorfic, for each gene there are different isoforms of alleles in a population, and co- dominant, both genes inherited by the mother and the father are expressed. The polymorfism can be a SNP (a single nucleotide is mutated) or it can invole hundred of base- pair. This means that the class I and class II molecules, which are more polymorfic than class III, can have a real different frequency in the population. THE COMPLEX In green we have the loci coding for the molecules beloning to the class II, in blue the one coding for class I molecules while in purple the one coding for class III. We can see in the image the locus of the complex on the chromosome 6, and starting from the centromer, there are class II, III and I. 00 We have more than 200 loci, so many genes, but all these genes are polymorfic. O Class 2 and I classical most important 3 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 All the molecules beloning to class I, are called HLA A, B or C; these are the 3 molecules that all our cells have on their surface. For class II instead, this antigens are call HLA DP, DQ and DR. PROTEIN STRUCTURE CLASS I: The HLA class I molecules belong to immunoglobuline super familiy, this means that they are very similary structuraly to the immunoglobulin, so to the antibody; in fact, they have this globular domain due to this surface bond inside the alfa-elic chain. pe The class I molecules have only one chain, one big protein chain that has a transmembrane 3Dstructure domain and a very short tail in the citoplasm, because they don’t have to send a signal. The class I molecules, because of this particular structure, need to bind another protein that is call beta2-microglobulin: this protein has a globular domain, a very short protein necessary to the perfec conformation of the HLA class I molecules. The antibodies have many globular domain such as MHC class I and II. CLASS II: The class II molecules are constituted by 2 chains: alfa and beta (class I have only one chain, the alfa chain). They are coded by different genes, they have 2 globular domain, a transmembrane domain with a very short intracellular tail. or normal protein 4 toShow the Ihre ofthey Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 BETA2-MICROGLOBULIN: In this image we see that the beta2-microglobulin is interracting with the alfa 1 and alfa 3 domain of the class I. no Poumorphye The beta2-microglobulin, differently from the alfa chain of the HLA molecule which is polymorphic, is monomorphic: this means thar everyone has the same beta2-microglobulin but different HLA molecule. Architecture of beta 2 microglobuline: it very short, it just present 100 aminoacids, and it intercat with the alfa chain. Without the beta 2 microglobulin, the alfa chain cannot be exposed in the cells surface. The beta2-microglobulin can reach and maintain the tridimensional structure; this is important not because it isn’t good having a collapsing molecule on the cell surface, but for the important function of the structure. Between the alpha I and alpha II domain there is what we call the pocket, in which, small peptides or what is inside, are shown on the cell surface.This is important to show what’s happening inside. This structure needs to be maintained, otherwise, in the collapsed form, the peptide cannot be exposed. Class I molecules are expressed almost in all cells with the exception of neuronal, endocrinal and some of muscle cells. This difference in the expression is a sort of way to repair from an eventually response of T cell against our antigen; if this happens against ephitial cells or tissue cells it is dangerouse because they provoce damage, but it is much more dangerous if the T cells kill the neuronal or endrocrinal ones. The expression of HLA molecules class I is constituve, but can be induced and increased on cell surface in response of some tissue factor for class II or citokynes. For example, the TNF or the interferon, which derive from the activation of toll like receptors, can increase the MHC molecules expression. As we can see in this image, there are a lot of class I molecules in our cell surface. They are all made by alfa chain and beta2-microglobulin. All these molecules present on our cells surface will jusr show some cell proteins, if they aren’t modified they won’t induce any kind of response. When our cells became infected, viral or bacterial protein start to be produced and they will be located on the pocket of the molecule. 5 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 We have different genes coding for HLA, HLB, HLC; all our cells have genes inherited by mother and fahter, two for HLA, HLB, HL → this is what we call polygenic. Inside the population these genes are different, we have more than 1600 genes for HLA, more than 2000 for HLB and more 1000 for HLC. This is why each of us has a different patter of HLA molecules on the cell surface; we have potentially bilions of combination. By the co-dominance features we can express the HLA, HLB, HLC from the father and form the mother at the same time, so every cells have 6 different class I molecules. 3dalla madre 3dalprove The set if the molecule is called haplotype. THE PRESENTATION Two globural domains, alfa 1 and alfa 2, generate what we call pocket in which something is shown outside, the red triangle represent the small peptide inside. The HLA 1 peptide complex is recognized pepure I specifically by the TCR expressed by the T lymphocyte, while the structure of what is inside pocket is recognized by the NK cells. When the NK cells patrol our body they feel the presence of MHC class 1 molecules; they only feel the presence of the HLA class 1 molecule, they are inibited because the HLA molecule interact with the inibitory receptor. While, for the TCR it is important not only the structure of the HLA, but also what is inside. So the TCR can specifically recognize what is inside. Thinking about a protein medium 1, 400 amminoacid can be degraded in very short peptides. 6 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 Class 1 present very short tail, constituted by 8/10 amminoacid. If we think to a very small protein, 400 amminoacid, you have a lot different peptides that will be shown by the HLA and that should be recognized specifically form different T cells and TCRs. The groove in the image (it is in the MHC class I) is not able to allow a peptide longer than 10 amminoacid to be inside. In this picture some of the amminoacid are not even in contact with the pocket. For the HLA class 1 what are important are two amminoacids which are called anchor amminoacids; usually, the amminoacid 2 and 8 should be for each kind of HLA class 1 a specific amminoacid. Different MHC class 1 bind different anchor amminoacid; this is important because in terms of human population, in the presence of different types of MHC class 1 and class 2 molecules, at least some of us will be able to show and activate an immunoresponse against the pathogen; this happens also with the new pathogens, for example with the coronavirus, some of us was able to face the infection without getting to much sick because the potency of the presence of different HLA class 1 and 2 in the population and so the presence of different TCR, allow us to face, potentially, any kind of pathogen. Going back to the groove of class I molecules, an important thing to remember is that any MHC class I protein can, actually, allocate, just because the presence of the same anchor peptide, different kind of peptides. Is not that one MHC class 1 protein can present only one peptide. The bond between the peptide and the MHC class I is not covalent, so these molecules can bind differnet type of peptides; this allow us to present different peptide of different proteins. NON-CLASSICAL HLA MOLECULES HLA E, HLA F, HLA G and NK are coded by genes inside this complex. They have a similar structure, so they are also bound to beta2- globulin, but an important feature is that they are less polymorphic than the classical ones. They are not the one presented usually what is inside, even if is not because HLA E present anyway some that is different from the classical peptide. Usually, they are not bound by the classical lymphocyte that we have in circle, the alpha- beta lymphocyte. We have two mains population of lymphocyte based on the type of receptor they present: the more abundant is alpha-beta population (80/85%), the other are call gamma-delta (the population present a gamma-delta TCR). 7  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