Natural Killer Cells PDF

Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 INNATE CELLS POPULATION PART 2 NATURAL KILLER CELLS “NK cells” means “Natural killer cells” and their function is to kill. These cells have a lot of granules inside, containing perforine...

Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 INNATE CELLS POPULATION PART 2 NATURAL KILLER CELLS “NK cells” means “Natural killer cells” and their function is to kill. These cells have a lot of granules inside, containing perforine and granzyme. The former is necessary to create pores in the membrane of target cells, while granzyme, which is a protease, is later used to activate other proteases, for example caspases which induce apoptosis in target cells. All the other populations of immune cells derive from, or belong to, the granulocytic/myelocytic population: they derived from a unique progenitor called the myeloid progenitor. The natural killer however, even if it participates in the innate immunity, derives from the lymphoid progenitor, just like lymphocytes, and they are morphologically very similar. Particularly, lymphocytes are quite similar to cytotoxic lymphocytes, which also have a lot of granules. Since NK cells are born to kill, they need to have some inhibitory receptors to switch off their activity; otherwise, all the weapons used by the immune system against intruders or non-self elements, can be used against our cells. Each receptor, to function, needs a ligand and, after the binding, they activate some cascade. Usually activated receptors switch on some kinase and the phosphorylation cascade leads to the activation of some transcriptional factors that by migrating into the nucleus, start the transcription of new genes. The inhibitory receptor, on the other hand, will activate some phosphatase. 1 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Education of NK cells in the bone marrow During the origin in the bone marrow the NK cells already express both activating and inhibitory receptors. The main function is indicated by which kind of receptor prevails. Usually, in a physiological state with no intruder pathogens, the inhibitory receptors prevail and the phosphatases switch off the activating receptors. The activating receptors will activate their killing function through the kinases and phosphorylation of the adaptors. In contrast, inhibitory receptors are able to maintain NK cells quiet through the activation of phosphatases, which eliminate phosphate groups from the kinases and switch off their pathways. When NKs circulate around our main tissues (or even in the blood), the inhibitory signals prevail because the inhibitory receptors are binding the MHC class I molecules. The MHC (or HLA) class I molecules are expressed by all nucleic cells: only erythrocytes don’t have the MHC class I (with some exceptions). The inhibitory receptor finds the ligands to switch off the NK function everywhere in our tissues. When the MHC class I is missing, or many other ligands for the activating receptors are exposed on the surface of the target cells, the inhibitory receptors are not activated at all and in that case, the signal coming from the activating receptors prevails. 2 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Some examples of receptors and their ligands: Important ligands: - Hemagglutinin => a viral component - MICA and MICB => they are usually exposed on our cells when metabolic stress occurs or even on neoplastic cells. They are very similar to MHC class I, but they don’t belong to the same family - Molecules from the HLA complex => they are usually bound by inhibitory receptors. MHC and HLA are the same molecule, but HLA is present in humans and stands for Human Leucocyte Antigen, while MHC means Major Histocompatibility Complex and it’s usually used for mice or other mammalians. These molecules are expressed on all normal cells and even on trophoblastic ones; this is very important during pregnancy to protect the fetal growth: since the fetus carries pathogens coming from the mother and from the father, he has different HLA that can lead to an inflammatory response. However, these cells do not actually express HLA (or they express another specific kind of HLA), so they are able to switch off the activity of NK cells. Cytokines which activate NK cells Cytokines are important to regulate the functional activity of NK cells. All the cytokines in the next page are produced by macrophages or dendritic cells (after the activation), or by helper lymphocytes. IL2 is also present in lymphocytes and it’s a sort of growth factor that helps this cells to proliferate. In NK cells it also increases lytic activity and therefore degranulation. 3 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 The major important cytokine for maintenance and proliferation of NK cells is IL15. Missing self In the left picture below, it can be seen a normal cell expressing the HLA class I that is binding the inhibitory receptor à NK cells are “quiet”. A damaged or infected cell can escape from the recognition of the immune system by decreasing the expression of HLA class I. In this case, when the HLA class I is missing, the inhibitory receptor cannot signal inside, so the signal coming from the activating receptors will prevail and the NK cells will degranulate the content and kill the target cell. The activation of NK cells caused by the absence of HLA class I is a phenomenon called “missing self”. The activating receptors recognize: - Stress ligands => present in abnormal cells; - Neoplastic cells; - Cells infected by viruses => especially by Cytomegalovirus or the Herpes Virus (decrease on the expression of HLA); - Cells infected by intracellular bacteria => they cause stress in our cells; - Cells covered by antibodies: ADCC. 4 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Antibody dependent cellular cytotoxicity Another process in which the NK cells are involved is the “antibody dependent cellular cytotoxicity” or ADCC. NKs have a particular receptor, the CD16, that recognizes the constant portion of antibodies; when this bond occurs, the activating receptor is triggered and the NK cells degranulate. NK cells are the major players for ADCC, even though some macrophages are also able to do it. This important mechanism is able to kill infected cells: the antibodies involved in the antiviral response are induced and bind the surface of the infected cells leading to their death. Even if the MHC class I is expressed, NKs can kill the target through the activation mediated by the antibodies. The activating and inhibitory receptors function in different ways because they have a different intracytoplasmic tail. The activating ones have the ITAM motif (intracellular tyrosine activating motif) while the inhibitory ones have the ITIM motif (intracellular tyrosine-reach inhibitory motif). These are very common even in other kinds of receptors, for example the CD16 also has it. The activator motif is reaching tyrosine that can be phosphorylated and function for the attraction points of kinases. The ITIM motifs are still reaching tyrosine, but when phosphorylated they are bound by a phosphatase. 5 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Cytokines which are produced by NK cells All leucocytes secrete cytokines. Even epithelial cells secrete cytokines, but leucocytes are the main producers. Different cytokines are able to activate different functions or different cells populations, but not at the same time: it depends on the pathogen or the damage they have to face. In case of viruses or internal bacteria infection, for example, macrophages and neutrophils are activated to phagocytes the infected cells. The NK cells and the killing lymphocytes have also to be activated and all of this is a sort of response that is activating by cytokines. Another example is: when we are infected by worms, we have to recruit eosinophil which are the only producer of major basic protein that are able to destruct the wall of the worms. To sum everything up: There are a lot of cytokines and growth factors that are mainly defined in different functions, such as the recruitment or the proliferation of immune cells, and are very important to modulate the immune response. These cytokines are usually produced after the activation of NK cells: - IL15 is important for a positive feedback mechanism: it maintain their activation and especially their proliferation. - GM-CSF is a factor that induce the bone marrow to produce neutrophils or eosinophils and monocytes. - INF-γ, which is very active on both T cells and macrophages. - TNF-α, which is also active in macrophages to maintain the M1 profile. This is really efficient in phagocytosis and in oxidative burst (and so in the production of ROS) because it creates a toxic environment that kills the pathogens. With the production of these cytokines, NKs can activate different kinds of cells, the inflammatory response and hematopoiesis. NKs are also able to kill both while circulating and extravasating into the tissue. Immune System - Natural Killer Cell - YouTube Natural Killer Cells (NK-92) Explainer Video - YouTube INNATE LYMPHOID CELLS Innate Lymphoid Cells (also known as LCNK) originates from the same lymphoid precursor as the lymphocytes and both produce cytokines. Despite this similarity, ILCs don’t have specific TCRs. There are 3 families of ILC: ILC1, ILC2 and ILC3. They differentiate to each other thanks to a certain kind of microenvironment produced by specific cytokines released by damaged or infected cells. When they differentiate in ILC1, 2 or 3 they produce specific patterns of cytokines, able to activate proper innate or adaptive cells in order to fight a specific pathogen. The gastrointestinal mucosa is full of so-called “innate lymphocytes” that are promptly activated and able to induce the inflammatory response. ILCs also activate the macrophages and, based on the cytokines they produce, activate the M1 or M2 macrophages: M1 kill pathogens and phagocyte them; 6 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 M2 participate in the end of the inflammatory response, so they secrete anti-inflammatory cytokines and growth factors to start tissue repair. They also trigger other functions from other types of cells, like the Goblet cells (which produce mucus) or Paneth cells (which produce antimicrobial peptides). Microbial signals ILC1, 2 and 3 differ on the microbial signal that activates the epithelial cells or the macrophages and dendritic cells residential in the tissue. ILC1 TNF induces ILC1, which produces IFN-γ; they are important for the activation of anti-viral and intracellular bacterial response. ILC2 The activation of eosinophils is induced by the ILC2 population, which will be differentiated in the presence of different cytokines. All these cytokines are produced by the epithelial cells, when the tissue needs to face big parasites like worms. Even in allergic reactions the binding with some molecules on our cells’ surface is able to induce the secretion of cytokines that activate the ILC2. The production of these specific patterns of cytokines (IL5, IL13), are also in common with TH2 cells, that are necessary to recruit eosinophils or to activate mastocytes. Allergic reactions may include sneezing or the contraction of the intestine mucosa (present in food allergies); these effects are due to the activation of mastocytes and eosinophils. ILC3 ILC3 cells are very common in the gastrointestinal epithelial gap and very similar to the TH17 population. It differentiates in the presence of the cytokine IL23, usually produced by Langerhans 7 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 cells. ILC3 produce IL17 and IL22, that are very useful for the fungal response (candida, aspergillus). Innate lymphoid cells - YouTube FIBROBLASTS Fibroblasts are abundant in the mucosae and the skin. They are used in many fields of research, such as tumor research and autoimmunity one. They produce a lot of metalloproteinases which are important in the inflammatory response because they destroy the extracellular matrix; doing so they open the way for the leucocytes that extravasate to reach the target area. Fibroblasts release also a lot of cytokines, therefore it is believed that these cells are also able to modulate and regulate the innate immune response. HEMATOPOIESIS There are 2 kinds of bone marrows: the red and the yellow one. Hematopoietic stem cells, which generate immune cells, are produced in the red bone marrow. It is characterized by a lot of capillaries (that are called sinusoids), hematopoietic stem cells, mesenchymal stem cells and adipocytes. Adipocytes, like osteoblasts, produce factors that are important to maintain the hematopoietic stem cells’ proliferation. During fetal life, hematopoiesis mainly happens in the liver and very little in the spleen. From the 4th or 5th month of pregnancy, it also starts to occur in the bone marrow. From birth on, the bone marrow is the only place in which hematopoiesis happens. After birth, the major hematopoiesis occurs in long bones, and, as the time passes by, it continuous in the ribs, sternum or vertebrae. 8 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 From hematopoietic stem cells, we can have a myeloid precursor or a lymphoid precursor. Thanks to cytokines and other factors produced by mesenchymal stem cells, adipocytes or osteoblasts, the myeloid precursor is able to generate all the innate cells such as: erythroid progenitor => differentiates in red cells; megakaryocytes => differentiate in platelets; basophil progenitor => differentiates in granulocytes, such as basophils and mast cells; eosinophil progenitor => differentiates in eosinophils; granulocytes-monocyte progenitor => differentiates in neutrophils and monocytes o when they extravasate, monocytes can differentiate in dendritic cells or macrophages. The lymphoid precursor can give T progenitor or the B progenitor. The T progenitor can generate the classical T cells, the NK cells as well as innate lymphoid cells. 9 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Hematopoiesis | Hematologic System Diseases | NCLEX-RN | Khan Academy - YouTube Single cell transcriptomic analysis Scientists were able to classify all the progenitors and mature cells with different morphological and functional features, thanks to the single cell transcriptomic analysis. Isolating all the cells present from the bone marrow at different ages and analyzing all their RNA molecules, scientists found out that there is a pool of stem cells which are able to give different progenitors. Each of the steps is characterized by a different profile; these progenitors can still differentiate in other cells and we know which progenitors are more similar to one other. These cells mature because of different patterns of transcription present in the nucleus or in the cytosol. However, the cells do not maintain these characteristics because they are genetically inherited, but because they have an epigenetic memory. Epigenetic modifications are reversible, therefore cells in the tissues are affected by the microenvironment: they have the potential to change à In a different contest they can change and differentiate in another type of cell; for example a macrophage can be M1 or M2 based on the cytokinetic environment; follicular lymphocytes can produce cytokines specific for TH1, but in a different cytokinetic environment they induce the production of TH2. à It’s important to remember that all immune cells are very plastic, so they can even change in the tissue based on the microenvironment. These changes are dependent on the kind of pathogens that the cells are facing. INNATE RECEPTORS Innate cells can recognize non-self elements due to the presence of specific receptors on the surface. These receptors do not recognize a specific sequence (in contrast with the adaptive response) but shared features or molecules on the pathogens’ surface. They recognize the “Pathogen Associated Molecular Patterns” or PAMPs, but they can also recognize the “Damage Associated Molecular Patterns” or DAMPs. Some of these components are only present in pathogens: Mannose or fucose residues, typical of microbes, bound to some protein; LPS or lipoteichoic acid => both are common in gram(-) and gram(+) bacteria’s wall; Double or single RNA coupled with DNA => present in our cells only when intracellular bacteria or viruses are inside them. Peptide bound to N-formyl-methionyl => this is typical of bacterial proteins, however some of our own proteins, in the mitochondria, are bound to formyl-methionine. These are part of DAMPs, because usually proteins from the mitochondria stay there and they never go out in the cytosol. When this happens, it means that the mitochondria are damaged, so the cell may be suffering. CpG sequences in the DNA => Bacteria have a lot of non-methylated CpG sequences. All these molecules, belonging to PAMPs and DAMPs, are recognized by some specific receptor called Pattern Recognition Receptor (PRR) or by soluble molecules called Pattern Recognition Molecules (PRM), such as C-reactive proteins and Pentraxin. These molecules can also be produced by epithelial cells (they are called acute phase proteins) and they are part of the physiological biochemical barrier because they are able to bind bacteria on the surface and then direct these bacteria to the innate cells to be phagocyted. Since it’s soluble, Pentraxin needs to be recognized by innate cells through CD89 receptor in order to activate phagocytosis. 10 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Danger signals: the alarmins Besides the component of coagulation cascade or the complement proteins, which are induced by the inflammatory response, there are a lot of molecules that can be recognized by our innate cells and activate them, just like PAMPs. Some examples are: - ATP; - [HMG-B1]; - Lipoproteins which can ingulf microphages or mitochondrial DNA; - heat shock proteins; Receptors present on immune cells Receptors that immune cells usually present on their surface: PRRs => PRRs are the most abundant receptors. They recognize all the molecules previously mentioned. o Some PRRs are exposed on the cell surface, some are free in the cytosol and others are expressed on the endosomal membrane; Cytokine and chemokine receptors; Adhesion molecules => they allow the cell to extravasate and adhere to the tissue; Hormone receptors => the immune system, in some conditions, functions differently in men and women, because it can be modulated by sexual hormones; Antibody fragments => for complement receptor; Scavenger Receptors => they recognize lipidic molecules, typical of bacteria or damaged cells; C-type lectin receptors => they recognize sugars on microbes, fungi, lipoproteins secreted by stressed cells; Toll Like Receptors (TLR) => they recognize PAMPs and DAMPs; Some types of scavenger, lectin and toll like receptors can also be present on the surface of macrophages and neutrophil and a lot of them can recognize all kinds of pathogens. The general function of all these receptors is to increase the rate or facilitate phagocytosis. After the recognition of a pathogen, they immediately activate some changes in the cytoskeleton of the phagocytic cells and induce invagination: the phagosome forms, it fuses with the endosome and it digests and eliminate bacteria. Some viruses or bacteria only survive when inside the cells, others can escape from the disruption operated by the lytic enzyme and live inside some endocytic vesicle. However even these endocytic vesicles have some TLR, so they can mediate the recognition of what is inside the endosome. DNA or RNA coming from damaged cells or viruses, present inside endosomal vesicles, can be recognized by Toll Like Receptors, and the killing pathway of these innate cells can be activated (the phagocytosis per se is not enough). 11 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Toll Like Receptors TLR has been discovered in Drosophila for the first time by Christiane Nüsslein-Volhard: she discovered that TLR in Drosophila induced the correct dose of polarization in embryos. I Later, Beutler and Hoffmann discovered that also mammalian cells had homologous Toll Like Receptors which were involved in inducing an inflammatory response. After that, it become known that plants, invertebrates and many other organisms have Toll Like Receptors which are able to defend them from pathogens. There are at least 9 TLRs in the human body even if the genes are 13. The extracellular portion is very similar to the Toll molecule in Drosophila. The small cytosolic tail is very similar to the one from the receptor IL1. As it is shown in the image below, TLR2, 4, 5 and 6 are expressed on the cell surface, while TLR3, 7, 8 and 9 are inside endosomes. It’s important to remember that, in order to function, TLRs need to either homodimerize or heterodimerize. TLR2 and TLR5 can heterodimerize: in this way they can enlarge the number of molecules they can recognize. In fact, in contrast with T cell receptors and B cell receptors, there are only 9 TLR, so there is a limited repertoire of recognition that can be increased by heterodimerization. TLR can recognize most of the pathogens (not worms) and they are expressed on most of innate cells, however sometimes they can be present on T and B lymphocytes. They can recognize DAMPs, so they are also involved in induction of some autoimmune reaction. They can recognize pathogens outside or inside the cell. 12 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 TLR: mechanism of action Many of TLR (like TLR4) are not able to directly bind the molecule present on the pathogen but they need a complex of molecules. In the image on the right, the LPS is not directly recognized by TLR4, but it’s firstly bound by a C-lectin called LPS Binding Protein and then by MD2, which vehiculate all these complexes to CD14. CD14 is a molecule mainly expressed on macrophages, monocytes and immature dendritic cells, but it’s also present on many of our innate leucocytes. After binding all this complex and LPS, CD14 is able to bind the TLR and activate it. When the TLR dimerize after the binding of the ligand, a signaling cascade has to be activated and this happens thanks to the TIR domain. The TIR domain is homologous to the cytosolic tails of the receptor for IL1, in fact TIR means “Toll IL1 Receptor”. After the binding of the ligand, the TIR domain is bound by an adaptor called MyD88: this adaptor is important because all the molecules previously mentioned can be affected by some mutation or misfunction that can lead to some consequences on the proper function of the immune system. MyD88 is quite common to all TLRs; it’s an adaptor which needs to bind the IL1 Receptor Associated Kinase (IRAK) that is usually associated with IL1 receptor. The phosphorylation of this kinase is important for the binding of TRAF6 (TNF-α- Associated Factor 6). After the binding of TRAF6, 3 different pathways, depending on the ligand, can be activated: 1) MAP Kinase pathway => this leads to the activation of AP1, a transcriptional factor which migrates into the nucleus and stars the transcription of new genes; 2) Activation of IRF7 (Interferon Response Factor 7) => this transcriptional factor migrates into the nucleus and starts the transcription of interferon genes; 3) NFkB pathway => this pathway induces the transcription of inflammatory cytokines and chemokines. NFkB is quite common to many pathways activated by immune receptors; its misfunctioning causes severe immunodeficiency. 13 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 One example is the mutation of the inhibitory kinase NEMO (this gene is present on the X chromosome) that causes a dysplasia where the main phenotype is high susceptibility to bacterial infection: since NFkB doesn’t work, it can’t activate the inflammatory response. People who are affected by this disease are characterized by rare and thin hair and conical or absent teeth. A CLINICAL CASE: DOUGLAS MOOSTER Douglas Mooster (6 years old) has pneumococcal meningitidis and frequent ear pneumococcal infection. He partially lost hearing and therefor his learning abilities are affected. He also has intestinal problems, scalp boils and skin infections, which are all responsive to antibiotics. This indicates that the child is very susceptible to bacterial infections. 1st step: Laboratory assay The purpose of the assay is to investigate the leucocyte count: - Normal T and B lymphocyte; - Monocytes; - Normal Complement function; - Normal antibody titers. The results show that Douglas has a normal leukocyte count and even a normal complex activation. For a successful diagnosis it is important to point out that Douglas was vaccinated against Streptococcus pneumoniae and Neisseria meningitidis, however his lymphocytes were purified and tested in the lab and they had no response: they did not proliferate or produce cytokines. The diagnosis is “susceptibility to pneumococcal infections”. What could be the cause? In order to test TLR function, leucocytes are stimulated in vitro with specific ligands of Toll Like Receptors, such as fucose, mannose, proteins, and viral DNA. TNF production is analyzed, because all activated innate cells produce TNF: it is one of the first inflammatory cytokine that is produced. Results show that TNF production was actually very low compared with a healthy person. 2nd step: stimulation with IL1 The low production of TNF indicated that the child’s cells are not well responsive. There are at least 2 reasons why the cells are not responsive: 1) Complete absence of Toll Like Receptors. However, in this case, there is zero production of TNF, not less, therefore this option is ruled out. 2) Dysfunctionality. Since this receptor has an intracellular portion similar to the IL1 receptor, the child’s leucocytes need to be stimulated with IL1. If they are responsive, it means that all intracellular pathways are working. If not, it means there are some problems specifically in the internal cascade. When stimulated with IL1, a healthy donor’s leucocytes activate MAP kinase and NFkB; for Douglas this doesn’t happen à This indicates that the adaptors, such as Myd88, IRAK and TRAF6, need to be checked. DNA sequencing demonstrates some missense mutation in the IRAK gene, therefore the internal pathway common in both IL1 and Toll Like Receptors is interrupted. With this form of immunodeficiency (presence of a truncated form of IRAK) there is still the recognition of the ligand by the TLR and the attachment of Myd88, but then TRAF6 cannot be activated. 14 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 In the image below there are all the potential combination for TLR, the molecular patterns that they recognize and the type of response. The activation of TLR mainly results in interferon type 1 production, and, in some cases, in the increase of MHC complex molecules expression. When cells recognize pathogens through their receptors, phagocytosis is increased, as well as the lytic activity and the production of cytokines to amplify (or start) the inflammatory response. 15 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 In this image, the barrier has been broken and all the bacteria are entering. The TLRs recognition induce the production of cytokines, that, thanks to a positive feedback mechanism, activate oxidative bursts and other killing functionalities of macrophages. Macrophages start to produce cytokines which recruit and activate NK and T cells that produce IL12 (which increases the killing activity of the NK). TH1 and NK cells both produce IFN-γ, which acts on dendritic cells by increasing the expression of MHC, and so by increasing the presentation of those antigens to the T cells and the immigration into the lymphoid organ. Maturation of Dendritic Cells The TLR signaling is very important to mediate the maturation of dendritic cells. This is increased by the recognition through TLRs and scavenger receptors. LPS, CpG, Double strand RNA and all the molecules reported below, are all good stimuli to induce maturation, which is the ability to activate the lymphocyte T cells. Dendritic cells will increase the expression of MHC molecules and other molecules necessary for the full activation of T cells. Another important effect is the change of expression of chemokine receptors on their surface: the immature dendritic cells express all the chemokine receptors that is necessary to recruit them on the site of infection. After the maturation they need to encounter the T cells, therefore they have to migrate in the lymphoid organ. Consequently, they need to change all the chemokine receptors expressed on the surface and expose only those that are responsive to the chemokines constitutively present in the lymphoid organs. 16  Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Errors in tuning TLR transduction In some cases, receptors can be too much active or activated too easily by self-molecules and therefor they can play a role in autoimmune reactions. One example is Chron’s Disease, in which TLR9 is too active. They can also be important for induction of susceptibility to shock syndrome, which can be due to a mutation in TLR4. I Some viruses try to escape the recognition by the immune cells, by decreasing the expression of TLRs. Some drugs, activate pro-inflammatory responses. On example is for example ImiquimodTM, which is used for HPV infection. This drug is an analogue of TLR7 and 8, it’s an oligonucleotide which mimics the RNA, activating the pro-inflammatory response when people are not properly activating the anti-viral response in case of HPV. Therapeutic exploitation of PRR o Some oligonucleotides, like CpG, are very important for maturation of dendritic cells. For this reason, they are used as adjuvant in many vaccines to increase the innate and adaptive response. If dendritic cells are more mature, they can activate T cells much more efficiently. The targeting and the inhibition of TLR9 is a strategy to delay or switch off the progression of an autoimmune disease (such as Chron’s disease). 17 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 CYTOSOLIC RECEPTORS There are three different types of cytosolic receptors that can recognize intracellular pathogens and bacteria:  NLR (Nod like receptors): recognize some peptidoglycans, like the N-acetyl glucosamine, which are exposed after the action of the lysosome;  RLR (Rig like receptors): recognize specifically the viral DNA;  CDS (Cytosolic DNA sensor): recognize the intracellular bacteria and the viral DNA. NOD family: The NLR family counts many molecules and receptors; the two most characterized are NOD1 and NOD2 that can recognize different sugars on the Gram (–) bacteria’s wall. These receptors have three different domains; the most important ones are:  the Nucleotide-binding oligomerization domain made up by some nucleotides’ sequences;  the CARD domain that is able to assemble another intracellular adaptor (not MyD88) that can bind and activate the pro-caspase1. One of their most important function, that occurs after the binding with the ligand, is to form a complex named inflammasome inside the cell: the inflammasome activates the caspase 1 (an inflammatory caspase) that cleaves the pro-IL1 and therefore release the active form of the IL1 and IL18. NODs are expressed in the immune innate cells but also in some epithelial cells (those that are more exposed to the bacteria) and mucosa cells (like the Paneth ones); in these cells the ligand can bind the NOD receptor and induce the secretion of potent anti-microbial peptides namely α-defensine and Pentraxin. In some condition, the NLR can be too much or less activated and therefore it leads to some autoimmunity reaction, as also observed for the TLR. The inflammatory response is not switched off in a proper way and because of that our cells can also damage, in absence of pathogens, our tissue. Thanks to the CARD domain (the same as the one present in the TLR), NOD2 by using the RIPK2 adaptor, can activate the NFkB pathway that leads to an inflammatory response. 1 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 Caspases, to be activated, have to be very close to each other because the activation process is allowed by their transactivation feature. NLRs, with their adaptors, permit this to happen. The active caspase1 can then cleave the pro-IL1 and allow the release of the active form of IL1. IL1 is one the first cytokines activated at the beginning of the inflammatory cascade but also IL18 is very important and involved into the inflammatory response. RIG like receptors: There are two main characterized receptors that are:  RIG (Retinoic acid inducible gene I);  MDA5 (Melanoma Differentiation-Associated gene 5). Both of them have two domains: the first one, the RNA-helicase domain, permits the recognition of the double strand RNA; the second one is that CARD domain that activates the Caspase1. They can interact with some adaptors inside the cells and stop the viral infections. Like the TLR3 and 9 (the ones that are in the endosome and that recognize the double strand RNA or the bacteria RNA), they activate the IRF transcriptional factors and induce the new transcription of interferon type 1. So the RIG receptors can heterodimerize after the binding and activate the IRF3/IRF7 transcriptional factor or, through the CARD domain, they can assemble the inflammasome with the releasing of the IL1 and IL18 from the cells. 2 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 Cytosolic DNA Sensors (CDS): CDSs are proteins that can recognize viral DNA in the cytoplasm and elicit anti-viral responses (like the transcription of interferon type 1) and autophagy (generation of an autophagosome, for example, around the damaged mithocondria). There are two main families of CDSs:  STING (stimulator of INF genes): active the transcription of INF1 and the autophagy mechanism;  DAI (DNA-dependent Activator of INF-regulatory factors): binds viral DNA and activates IRF3 leading to INF1 production. A summary of what we have seen until now: The interferon system: Cytokines are mediators that allow the communication not only between immune cells but also between immune cells and epithelial/mucosal ones (for example, those belonging to the mucosa). There are three types of interferon:  Type I is represented by INFα and INFβ;  Type II by the INFγ;  Type III by the INFλ, INFω and INFφ. All the interferons bind heterodimers receptors that are composed by an α and β chain. The type two receptors are formed by two dimers: after the binding of the cytokines, the α/β - α/β chains dimerize. These receptors don’t have a kinase activity themselves but they need to be bound, in the cytosolic tail, to a kinase called JAK or TYK. The phosphorylation of the cytosolic tails allows the dimerization 3 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 of STAT2/STAT1 (for INF I and III) and STAT1/STAT1 (for INF II). This complex can be bound to some other factors, but the most important thing is that it translocates into the nucleus in order to modulate the activation of some genes that are active in fighting viral infections. The INF type I and the dimer STAT1/STAT2, also called ISGF3,when activated, binds the IRSE (interferon sequence responsive elements) that can start the transcription of different genes such as mx1, oas, pkr, Irf7. Type I receptors is often induced by TLRs and cytosolic ones which recognize double strand RNA and other pathogens’ molecules. The INF II forms the GAF complex that binds the promoter of Gas (gamma activating sequence) and induces the transcription of some genes, such as ip10, mig and irf1, that are chemokines and therefore are involved in the attraction of other molecules. INF type I receptors are also expressed on epithelial cells which can directly fight the infections by using some enzymes:  Oligoadenylate synthetase: it’s an enzyme that polymerize the ATP and when it is inserted on the viral RNA, this is sensed like a break or damage in the molecule that will be degraded.  PKR kinase: it is a protein that phosphorylates the elF-2 factor that usually regulates the protein synthesis. When it is phosphorylated, it inhibits the translocation and the induction of the host machinery that synthetize the viral proteins.  Mx1 protein: it inhibits the proliferation and replication of genetic viral material (especially the influenza one). A summary of the receptors we have already discussed: 4 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 Cytokines and chemokines Communication codes within the immune system: There are many hormones, like corticosteroides, adrenaline and noradrenaline, that regulate the activation or the inhibition of the immune cells. Cytokines (i.e. interferons) can be produced by both endothelial and immune cells and activates the immune response as an alarm signal. Cytokines begin their function when bound to a receptor. Most of their receptors are inducible but they can also be constitutive. HLA antigens by binding to the TCR, allow the communication between all cells and the immune ones. Membrane ligands can be expressed also by the antigen-presenting cell and together, they fully activate the lymphocytes. Cytokines and chemokines: Cytokines and chemokines belong to the same family but because the latter mediates the chemoattraction, they have been called chemokines. Both of them:  Are messengers that can activate the adaptive and innate response of the immune system;  Can work as SOS by regulating the hematopoiesis and therefore the amount of circulating immune cells that are needed to fight a specific pathogen;  Favour the extravasation: for example, the TNF cytokines, that are active on the endothelial cells, allow the rolling first and then the strict adhesion of the leucocytes; these kinds of molecules change their conformation to become more similar to those expressed on the glucosides thanks to the releasing of cytokines.  Can block the intruder spreading by inhibiting the viral replication and/or packaging, activate fibrin deposition and the blood coagulation cascade (these last two strategies are very important). Cytokines are secreting molecules that to function well, need to bind a specific receptor; if this doesn’t happen, the general activation of too many lymphoid cells can damage our tissue. 5 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 There are many families of cytokines:  Interferons: INF-α, INF-β, INF-γ,…;  Interleukins: IL1, IL2, …, IL37;  Tumor necrosis factors are soluble molecules, active in the endothelial cells, that mediate the necrosis of tumor cells by disrupting the blood vessels; TNF-α is immediately released after the recognition of the pathogen by the PRR.  Colony stimulating factors: G-CSF (granulocytic –CSF), GM-CSF (granulocytic and monocytic –CSF), M-CSF (macrophage –CSF). The first two works to induce the hematopoietic stem cells to differentiate in their precursor (basophil and mast cells//neutrophils and monocytes). The M-CSF doesn’t work on the bone marrow but in the tissue because it induces the differentiation of the monocytes in macrophages. They are very similar to growth factors and hormones.  Transforming growth factors: TGF-β (the TGF-α is not a cytokine but a real growth factor for the epithelial cells); it is one of the most pleiotropic cytokines that usually is released by suppressive cells  it is an anti-inflammatory cytokine. Main features:  Cytokines are so small (17-20 kDa) that if you are performing a WB you have to be careful because they can exit from the bottom.  They work in dimers, rarely as monomers, and can have different structures for similar functions and viceversa. This is a very important advantage for us because if one of them doesn’t work, there will be always another cytokine, with a similar function, that can compensate the loss of the previous one.  They have a short half-life (minutes/hours). o They can be neutralized by some inhibitors circulating in our blood that impede the recognition with the receptors; o They can be very quickly catabolized by kidneys; Cytokines’ storm (maybe you’ve heard it correlated with coronavirus): the cytokines are not immediately catabolized and therefore they can start the iper-inflammatory response in many tissues at the same time.  They are pleiotropic: the same cytokine can signal different kinds of cells and the same cell can be affected by different cytokines.  Cytokines can synergize (initiate the same function together) or antagonize with other cytokines  there will be an algebraic sum of the two effects; in some cases, one can repress the signal of the other cytokine (INFγ and IL4). Cytokines can work in an autocrine way (the cell that releases the cytokine is affected by the same cytokine) or … … in a paracrine way: cytokines are released from the immune cells activated by the recognition of a pathogen by 6 Alessia Gecchele / Larisa Laios Lesson 4 – Immunology (Paola Cappello) 14/10/2021 the PRR; they can act in a paracrine way with all the surrounding cells but only those with an inducible receptor can be activated. Another specific feature of cytokines is the polarized stimulation: In the lymphoid tissue, during the antigen presentation, from a dendritic cell to a T cell, an immunological synapsis (composed by two immune cells) is formed: cytokines are secreted in a polarized way and this allows only specific T cells to use this cytokine. The dendric cell can bind at the same moment different T cells but all of them have different TCRs; the polarized secretion allows only the T cells specific for that antigen to be activated by those cytokines. This is fundamental for the starting of the clonal proliferation and for the differentiation in different subtypes. Endocrine stimulation (like hormones): this happens only for the colony stimulating factors because they have to affect the hematopoietic cells and so they have to travel long distances. Cytokines’ receptors features:  Highly specific: their dissociation constant is very small (10 -11M);  Temporarily expressed by a few cells and only in the right moment; they are induced by a special signal or can be constitutively expressed.  Only a part of the receptors is always present: most of them are heterodimers, trimers,… and so, out of three chains, only one or two can be constitutively expressed (the other ones are only induced in the right moment). Main receptor families: In the common γ chain: receptors can be heterodimers or especially heterotrimers and all of them are composed by the same γ-chain; the other part of the receptors, is a specific chain that will recognize specifically the IL. The same is for the family of the common β chain: the β one is the same and the α is specific and different for each cytokine. The TNF receptor is a homotrimer kinase, made up of three identical chaines that join together after the binding of the TNF ligand. 7