Immunology Lesson Notes (PDF)

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...

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. 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:  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 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 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 COMMUNICATION CODES 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 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) 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 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. 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. The problem for the organism was how to catch all the microbes inside our cells. The human antigen is the HLA and is ha the sam meaning of MHC. The difference is that MHC is for all the speacies, while the HLA is only for humans (= human leukocyte antigen) 2 Francesco Grossi / Michelle Guichardaz – Lezione n°6 – Immunology (Prof.ssa Cappello) – 21/10/2021 - 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. We have more than 200 loci, so many genes, but all these genes are polymorfic. 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. The class I molecules have only one chain, one big protein chain that has a transmembrane 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. 4 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. 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. 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 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 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