Lesson 20: Immunology - Autoimmune Reactions PDF

Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 Autoimmune reaction Autoimmune reactions are strictly related to hypersensitivity reactions as we have seen in the past lesson (most of the hypersensitivity reac...

Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 Autoimmune reaction Autoimmune reactions are strictly related to hypersensitivity reactions as we have seen in the past lesson (most of the hypersensitivity reactions – type 1,2,3,4 – are also responsible for the induction of autoimmune diseases). What is an autoimmune response or reaction? It is the immune response against the self-antigen and so against own cells. The first one to describe this kind of immune response was Paul Enrich in the early ‘900, which actually described this type of reactions like an “horror auto-toxic”. At that time the knowledges about immune system were very few, indeed the macrophages and microbes had just been discovered, but he realized that in some individuals, sometime, something of their property, the immune system, was reacting against the liver, the kidney, or other organs. The first experimental and clinical evidence were only in 1950 and the first two clinically recognized autoimmune disease were the lupus erythematosus and the multiples sclerosis in the 1960. We have to keep in mind that to state that a disease is an autoimmune disease, it is necessary that the autoimmune reaction generates also a tissue damage and therefore clinical symptoms. Every day we can experience some autoimmune reactions, but without any kinds of consequences in terms of tissue damage and so clinical symptoms (none autoimmune disease). à A pathology, to be a stable established autoimmune disease, has to present also a self-tissue damage. Until now we have about a hundred autoimmune disease recognized. Noel Rose, the father of the autoimmune disease The scientist was working in experimental models, using rabbit and mice, to describe what then has been called Hashimoto’s disease, that is a thyroiditis. He was observing that the presence of antibodies in mice were because of this thyroiditis, but when he submitted the paper, it was rejected: it was claimed, indeed, that there wasn’t so much evidence in what he was observing also in clinic. He continues to work until he was able to describe this pathology also in humans. Autoimmune reactions can be related to the presence of autoimmune antibodies or T cells. When the autoimmune diseases are induced by the presence of autoantibodies, the mechanism at the base can be due to the triggering, by the autoantibodies, of some molecules, or type 2 and type 3 hypersensitivity can occur. (The type 1 isn’t relating to autoimmune disease but to allergies). The presence of autoreactive T cells usually induces the hypersensitivity type 4 and tissues damage, that is at the base of autoimmune disease. 1 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 The autoimmune disease can be local or systemic based on clinical symptom: if the autoimmune reaction is local, it is confined in a specific tissue or specific organ, while if it is systemic, it involves multiple organs. Why and how does autoimmune disease arise? All these 4 conditions have to be performed: 1. An autoreactive T cell clone must exit from the thymus (escape from the central tolerance and so from the negative selection) and should be released in the periphery. 2. Even in this case, this T cell needs to encounter the antigen, and this is not so easy because many self-antigens are ignored by our T cells; some metabolic arrangement or some progressive tissue damage need to be present in order to increase the amount of the self- antigens that can be representative and more present on the cell surface of antigen presenting cells. 3. The third mandatory condition is that the peripheral tolerance mechanisms must fail thanks to the presence of Treg cells, CTLR4, anti-inflammatory inhibitory cytokine released by Treg, tolerogenic dendritic cells (that have been induced by the presence of the regulatory T cells) and IL2 sequestration. 4. The autoreactive clone, inducing maybe also by B cells and so the effector mechanism can be T or B dependent, must create tissue damage. Usually, the autoimmune disease related to the failure of central tolerance are rare because it is quite strictly controlled, and we know very well the mechanisms. There are only, at now, two autoimmune diseases known to be related to the absence of AIRE; this is a transcriptional factor of the medullary epithelial thymic cells which is necessary to self-peptides that can be not only the thymic proteins (these are also expressed by the cortical epithelial) but also tissue specific proteins (like the insulin or the troponin) and all those proteins that are actually specifically related to other type of tissues. The lack of AIRE induces the APS (autoimmune polyendocrine syndromes) or the APECED (autoimmune polyendocrinopathy whit candidiasis and ectodermal dysplasia); these syndromes are active against many organs, especially glands, and cause important consequence related to metabolism of these hormones. All the other autoimmune diseases are mainly due to defects in the peripheral tolerance mechanisms such as: The presence of an inflammation, also a chronic one: the presence of a continuous inflammation, even at low intensity, is required. The molecular mimicry by pathogen antigens: some peptide from antigens are very similar molecularly (the aminoacidic sequence is very similar to the one of self-protein). Defects in some of the component of the immune system, such as dendritic components or the T, B, cytokines or complements and so on. Epitope spreading. 2 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 Why can inflammation increase the risk of autoimmune disease? During inflammation, many danger signals, such as the alarmins and the DAMPs, are released and this triggers the activation of the APCs. When there is an infection, the phagocyte are activated in this case by the PAMPs and they will migrate to the lymphnode where they will activate also some autoimmune autoreactive T cell clone, sometime, just for the bystander effect: the cytokines released in this milieu, because they are very contact to contact, allows the activation of the neighbour T cells in the lymphoid organ, even if not efficiently like the specific T cell clone. In particular during the inflammation, specifically after an infection, you never have the activation of regulatory T cells because the native T cells usually are autoreactive and recognize the self-antigen and not the pathogen antigen related; In this case the pro-inflammatory cytokines impaired the Treg function that means the Treg release of inhibitory cytokines such as TGFβ, IL10, IL35. The inflammatory response due to a strong infection is mostly a good soil for the induction of an autoimmune disease; experimental evidence is shown in the image: This is a tissue from the synovia, where synovial space in the arthritis or normal joint can be observed. It can be seen that during the autoimmune disease inflammation (in the left image, the stained cells are macrophages and dendritic cells), if you target the dendritic cells, and so you eliminate the accumulation of dendritic cells, the symptoms are getting better. à This means there is a direct relation between the presence of dendritic cells in the joint and the appearance or the progression of the symptoms. This happens because, usually, the inflammation release danger signals that activate the dendritic cells or plasma cyto-dendritic cells, based on the pathogen, and when the T or B cells are activated, and the production of antibodies is increase, we will have the tissue damage and the release of self-nucleic acid or self-protein; these, in the inflammatory context, can be presented as a pathogen antigen from the dendritic cells and so can activate, in turn, others autoreactive T cells. 3 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 With viruses (many autoimmune diseases are triggered by some virus infection), the most important cytokine released is the interferon type 1 and the interferon gamma (released by the macrophages activated form the interferon type 1). With the enter of the virus in the cells, in the image above we can observe an epithelium, the viral antigen became more representative on the epithelial cells thanks to the up regulation of MHC class 2 and 1 due to the presence of interferon. The overexpression of this peptide-HLA complex induces the recognition by the CD8 cells that of course will secrete perforin granzyme, cytokines such as interferon gamma that activate the NK cells and macrophages. In general, cytotoxic T cells will destroy these infected cells, but by killing them of course also our cells will be damaged; they will release other kinds of self-antigen that can be captured by the B cells, macrophages or dendritic cells that are in the tissue and, thanks to INFγ, will increase the presence of those macrophages and B cells that do not have such a good expression of HLA class 2. à they will increase class II molecules, therefore the activation on CD4 T+ cells, that will release cytokines and activate more cytotoxic autoreactive T and B cells against these self-antigens. The B cells, finally, will produce autoantibodies active against our protein. Molecular mimicry The molecular mimicry stands for the similarity of peptide from pathogen and self-antigen. In this table, different and autoimmune pathologies like the rheumatoid fever, the multiple sclerosis, the intestinal inflammatory disease (that is very similar to bowel disease) are represented and the comparison from two peptides, one coming from the self-protein and the other coming from the pathogen is shown. 4 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 In rheumatic fever, for example, people show T cells reactive against both the human myosin and the streptococcus wall because the two proteins shared many ammino acids in their sequence, especially in the small epitope (so the 8/9 epitope), that are presented in HLA class I recognized by the cytotoxic CD8. Sometime, also the anchor ammino acids (the ones that are necessary for HLA class I) are involved. This theory it’s corroborated by the presence of T cells, from the individual, that in vitro recognize self-antigen and pathogen, but at now there are no evidence of a direct relationship about the two clones. The theory simply states that when you have the normal presentation of epitopes from pathogens (like in this case), some autoreactive T cells, that have the TCRs able to recognize the similarity of the pathogen epitope, even if is specific against the myosin, are activated; , the T helper activated cells release cytokines and switch on autoreactive B cells and CD8 cells, which in this particular moment will react against the infected cells by the streptococcus. The problem is that these cells remain active and therefore they will also encounter the real antigen on the normal cells and react against it. à after the clearance of the pathogen, and so the end of infection, they will continue to find the antigen expressed and induce the tissue damage. Abnormalities in compartments of immune system - If we lack of FoxP3, we will be affected by IPEX, a syndrome that involve many endocrine tissues but also the intestinal ones: if we don’t have regulatory T cells we will experience in autoimmune disease. - Abnormalities in antigen presenting cells: in rodents (not yet in humans) it has been demonstrated that when dendritic cells are triggered (activated by alarmins or PAMPs), for some reason they are already matures and so they express co-stimulatory molecules, cytokines in a more abundant way compared to the normal individual. In this case, mice physiologically develop intestinal inflammation. - Abnormalities related to B cells: there can be many mutations that can lead to autoimmune defects in B cells like, for example, the absence of Fas, a death receptor. B cells can be killed by a cytotoxic cell, that express a Fas ligand, or a T cell after the activation and this is called fratricide because other activated T cells expressing Fas can be killed. When B cells lack of Fas, they cannot be killed at the end of an immune response and so we don’t have the decline of B cell activation and these B cells will continue to produce antibodies; The presence of too much antibodies is dangerous because they can perform immunocomplexes that will induce the destroy and the injury of the tissues. 5 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 - Abnormalities in conventional T cells: for the antigen presenting cells, for some epigenetic reason, in some tissue, T cells remain active after an infection and ready to secrete perforin and granzymes (if they are CD8) or cytokines (CD4). In this case they remain like if they are already primed, the same as memory T cell, and ready to induce the tissue damage. - Abnormalities in cytokines: In many autoimmune diseases it has been found elevated the level of a specific cytokines, based on the pathology; the increase of IL12 induce Th1, interferon gamma and the activation of more macrophages that will release TNF, a death cytokine that, especially in epithelial cells, will bind to the TNF receptor (death receptor like FAS). In other cases, the increase of IL6, TGFβ and/or IL23, will induce Th17 that is very powerful against fungal infection but, by triggering many cells like fibroblast, chondrocyte, osteoclast, it is very dangerous for our cells; it induces IL17 and especially IL6, a pro-inflammatory cytokine, involved in multiple sclerosis. In the lupus IL15 and IL27 increase; à even the cytokines have an important role in autoimmune diseases because they are responsible for the connection of the innate and the adaptive system and the activation of the cells. In some cases, there are some clinical evidence that these cytokines are very important: if you treat this patient with an antibody against the cytokine involved, you can have a relive and have a melioration if the symptoms. In the rheumatoid arthritis, the use of TNF meliorates the symptoms, but after a prolonged treatment, the patient worsens like if the autoimmune response is exacerbating instead of being dumped. - Defects on complement system: the C3, when it’s lacking, induces some autoreactive disease or autoimmune reaction because the C3b, the portion of C3 complement component, is necessary for the clearance of immunocomplex: the C3b also bound the F portion of the antibodies that then is recognized by red blood cells and transported to the liver or spleen for the degradation. It has been demonstrated that for some reason the immunocomplexes with the pathogen antigen are not long lasting: after the clearance of the pathogen are destroyed by the transportation to the liver or spleen. On the other hand, the immunocomplex with self-antigen seems to be long lasting; the reason can be that the pathogen antigen disappears after the pathogen elimination while the self- antigen is continuously present. Some self-antigens can be internal released only when the tissue is damaged, so even some self-antigens are not so present or available for binding antibodies This is maybe due to some defects in the activation of the complement because, during the pathogen infection the antibodies bind the pathogen antigen and efficiently activate the complement with the removal of the complexes. During a self-antigen release due to few cells, there is not a big inflammation like during the infection, and therefore the complement is not efficiently activated: the immunocomplexes, without the binding of C3b, are persisting in circulation, they can induce the type 3 hypersensitivity and amplify the tissue damage. 6 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 Epitope spreading Sometime during the immune response against a specific cell type, we have the activation of other T or B cell clone that recognize the so called cryptic or hidden epitopes: usually, they are not seen and only when epithelial or endothelial cells, or other types, are destroyed, they become visible to the immune system. An example: The pemphigus is an autoimmune disease in which there are a lot of blisters (vesicles) initially in the mouth there is a progression and individuals also develop vesicles on the skin. Initially the mouth blisters are induced by the recognition, from the antibodies, of the α-desmoglein3, that is a specific protein present in the buccal mucosa; the antibodies induce the complement activation and the destruction of buccal mucosa. By destroying these cells, another antigen, very similar but more abundant in the skin, the α-desmoglein1, is released and it activates the production of antibodies against it that will induce vesicle where the antigen in more abundant. What we have seen represents the epitope spreading: the reaction against a specific antigen, amplify the T and B clone activation against other antigens. This means that the number of epitopes after the first reaction is increasing, and it is activating against other antigens. The same happens also in diabetes type 1: we have lymphocytes against the GAD (glutamic acid decarboxylase), an enzyme expressed by the beta cells, and from the destruction of these cells other T cell clones are activated against the insulin and other their proteins. Let’s see now the pathogenetic mechanism: Pathogenetic mechanism means the effector mechanism that induce the autoreactive disease. When cells are destroyed, in pemphigus for example we don’t know exactly why some B cells are active against the desmoglein 3, antibodies against this protein, that is very abundant in the mouth, are produced. By destroying these cells of the mouth, the release of other antigens, that are usually low expressed and not visible by the T cells, is allowed; the release happens in presence of inflammation: the protein can be now well presented to the other T-cell clone, and this soluble protein is now available for B cell recognition; this is allowing the activation of other clones, specific for this one that is more abundant in the skin and therefore can induce the destruction of keratinocytes and so the blisters. This happens because in normal condition the desmoglein 1 is like the other self-antigens and it is not so well representative; even if it’s expressed on MHC class 1 and 2 on the cells surface, it is not in an inflammatory context and so it cannot activate the dendritic cell for the presentation and so on. This is only to mention that during an autoimmune reaction, more autoreactive T cell clones are activated and usually they will be ignoring this self-antigen and be active against a foreign antigen. 7 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 The effector mechanism: The presence of antibodies can activate the phagocytosis or induce, meliorate and amplify the phagocytosis; this happens in some cases of thrombocytopenia autoimmune disease because their induction creates an hyperreactive hypersensitivity reaction of type 2. The presence of antibodies can also activate the C3b and again induce the phagocytosis. This happens, for example, for platelets and for all the antigens that are bound to this surface and not soluble. If they are soluble, second image, they activate the hypersensitivity reaction type 3 and so the activation of the complement and the formation of the mark like in the haemoglobinuria. Many types of anaemia are induced by the presence of antibodies against the red blood cells and so the activation of the complement. Others autoimmune disease are activating by the presence of antibodies that specifically target some specific molecule (without any cell/tissue injury); this happens in the thyroiditis where both types (GD and TH) are induced by the presence of an antibody against the receptor of the thyroid hormone. In this pathology the function of the receptor is amplified inducing an increase in releasing of the hormone; In the myasthenia gravis, the antibodies induce the endocytosis of the receptors and so their loss of function. 8 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 Some examples of autoimmune disease: The thyroiditis exists in two different forms with opposite clinical effects due to a different pathogenic mechanism: one is the Graves’ disease (GD) and the other one is Hashimoto’s Thyroiditis (TH). The first form is characterized by high level of hormone (tirodoxin); the other case with low level. The clinical symptoms are also different: hyperthyroidism compared to hypothyroidism, tremor, tachycardia, weight loss compared to depression, fatigue weight gain. Even if the symptoms are opposite, in both cases we have autoreactive antibodies against the receptor and some autoreactive T cells (these autoantibodies are IgG, means that the CD4 cells have been activated). In this image, the thyroid with the follicles, that are the one producing and containing the hormone, can be observed. In a physiological situation, left image, thanks to the activation of the receptor, the hormone is released and can circulate. In Graves’ disease, the autoantibody against the receptors makes them constitutively activated therefore there will be an increase in the concentration of the hormone resulting into hyperthyroidism. On the other hand, in Hashimoto Thyroiditis the autoantibodies mediate a different mechanism that is the reduction of the function of the receptor resulting in a decrease of the hormone release. In addition, in Hashimoto disease, the antibodies against the receptors will also activate the phagocytes and especially the NK cells: the FC gamma receptors CD-16 on the NK cells are triggered by the antibodies and this bound will induce the de- granulation. 9 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 To sum everything up, the Hashimoto disease also include the partial damage of the thyroid follicle due to the fact that cells are killed by the NK. Myasthenia gravis Myasthenia gravis (MG) is another example in which autoantibodies are directly mediating the effects through the antigen binding site. It is a neuromuscular disease where the neuromuscular plaque is the target of the autoantibodies; this induce a weakness that can be at vary degrees with a wide range of symptoms including the loss ability to contract some muscles. In some cases, the ones involved can be the facial muscles resulting in people which are no longer able to speak, in others, problems connected to diaphragm or the muscle responsible of the respiration can be fatal due to respiratory insufficiency. Which are the mechanisms of action? The presence of autoantibodies against the receptors, induces their capping and endocytosis resulting in muscle cells no more able to respond to the acetylcholine due to the lack of the receptors. Even if the neuron cells are perfect in releasing the acetylcholine, the muscle cells cannot contract themselves and sense the presence of this neurotransmitter. In this case the recruitment or involvement of the innate cells is not required. Lupus The lupus disease is named after the presence of skin lesions on the face that reminds the face of a wolf. This is due to the presence of immunocomplexes that induces the activation of the complement, especially at the junction dermo-epidermal and so not only in the face, but also in other region of the body can be present these kinds of lesions; glomerular cells in the kidneys, for example, are very susceptible and destroyed by the activation of the complement due to the presence of immunocomplexes. 10 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 Also T-cell can be effectors and be responsible for the pathogenetic mechanism of some autoimmune disease: they can directly kill, if the autoreactive cells are CD8, the tissue cells and induce the injury, or release the cytokines that can activate the CD8 or the innate cells. In this table, there are examples of autoimmune disease induced by the T-cells activation against some antigens (multiple sclerosis – due to the presence of Th17 –, the murine autoimmune encephalitis – that is the same of the multiple sclerosis in human –, diabetes,…). Diabetes There are two types of diabetes but only the first is an autoimmune disease (the second type usually arise during the adult life). In type 1, there is no production nor response to insulin, so the level of glucose in circulation rise resulting in excessive hunger and thirst, increase urination, weight loss,…. The mechanisms that induce the type 1 diabetes involve macrophages and T and B cell, especially T cells activated against the beta cells in the island of the pancreas; in all type 1 patient, antibodies and T cells against many of the enzyme, like GAD or phosphates and even against insulin, expressed by these cells, have been found. The other evidence is that, when it’s possible to analyse the pancreas of these patient, there are a lot of lymphocytes infiltrating in the pancreas and the beta cells, in the presence of diabetes, have the peculiarity to express the MHC class 2 molecule. These cells are not among the ones that can express MHC II, unless they are in particular situation like in the presence on interferon gamma. These cells, in the insulae, are the one that are triggered by the autoreactive T cells, so even if the gamma and alpha cells are producing glucagon and somatostatin, the beta cells are the only one that are killed. Even if we know for sure that the pathogenetic mechanisms are due to aberrant activation of the immune system, we still don’t know which are the specific one. 11 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 It has been demonstrated that the beta cells, represented in the image, in a patient or in an experimental model, at a certain point, start to release a lot of chemokines, specifically CCL2 and CXCL10, that induce the recruitment of neutrophil and monocytes (that become macrophages in the tissue) and specifically TH1 and CD8 T cells. Probably are the β cells themselves that induce the recruitment of the immune system, starting to express the stress ligand MIC-A and -B, that are recognized by the activating receptor of the NK cells; these cells are then destroyed, and due to the epitope spreading, the recruited T cells, thanks to presence of macrophages and their ability to present the antigen, we can have the activation of other T helper and cytotoxic clone against these beta cells. One evidence, that has been reported in many patients with type 1 diabetes, is that there is a mutation in the DQβ1 chain (so in the HLA class 2 molecule); in this case instead of having the aspartate there are the valine, serine and alanine. This mutation changes the pocket of this molecule and so two possible scenarios can happen: - the peptide responsible for the diabetes, the one that then can activate the autoreactive T cells, cannot be presented and, in the thymus, there won’t be the negative selection of autoreactive T cells. - the presented peptide activates too much the escape of autoreactive T cell in the periphery. It has been demonstrated that this mutation is not enough to induce the type 1 diabetes: it’s true that many patients have this mutation, but this isn’t enough to cause the pathology. 12 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 What are the variables and determinants that can affect autoimmune diseases? 1. Genetic predisposition; 2. External triggers (chemical agents or infections); 3. Hormonal influences. Of course, some genetic predispositions are necessary but external triggers are also important because having a gene is predisposing but not enough to induce the disease. The genetic predisposition will enhance or will favour the induction of self reactively lymphocytes. For example, in the presence of an infection, there is the activation of the antigen presenting cells that will activate the specific pathogen and the specific T cells but also the self-reactive lymphocyte and their expansions. Some of these genes, that can increase the risk of autoimmune diseases, are related to the HLA complex class 1 and 2. Only B27 has been related at 100% to an autoimmune disease, the ankylosing spondylitis, because all the patients have this allele. The other genes, second table, aren’t associated with HLA but they belong to the cytokines, to FC gamma receptor, CTLA4, … 13 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 In the table some chemical agents and pathogens associated to some autoimmune disease are shown; for the chemical agents there’s no direct evidence proving that by using or by being exposed for long time to them you will develop the disease. For the viral or bacterial infection there is no direct evidence, but we can affirm that due to molecular mimicry, they are really favouring the induction of autoreactive T cells. The lupus, the herpes, the viral infections, and also the one that are chronic, are responsible for the activation of B cells and so the presence of a lot of antibodies. Concerning the hormonal influence, we’ve already mentioned that women and men react differently to different stimuli and if it’s good for women to have an intense response against many infectious agent, is not so good for the autoimmune diseases. As you can see in the table, 80% of people affected by autoimmune disease are women, so women are preferentially affected by autoimmune disease, except for the uveitis and the ankylosing. This is because sex hormones influence the immune reaction but also glucocorticoids, that are stress hormones, by inhibiting a lot of function, have an anti-inflammatory role. 14 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 When the metabolism of glucocorticoid is altered for some reason, some of these brakes are lost resulting in an hyperactivation of our immune cells, tissue damage and autoimmune disease. The conventional therapies for autoimmune diseases are all targeting the inflammation, so anti- inflammatory or immunosuppressive drugs are used. Another approach is represented by the plasmapheresis that clear all the autoantibodies reacting against self-antigen. The immunotherapy Immunotherapy tries to interfere with the mechanism that generate innate and adaptive responses. The aim is to inhibit the response to self-antigens, but not the general one of the immune system because, in this case, people that are immunosuppressed will be more susceptible to infections and not harmful pathogens can become very dangerous. à So, the immune therapies want to inhibit the specific response against the self-antigen, in order to cure the disease without affects the response to other pathogens or the onset of tumours: the immunosuppressive agents are also inhibiting the immune response that neoplastic cells can induced and therefore they can be recognized and destroyed before giving the clinical mass. The prospective are to induce a stable tolerance against the self-antigens in different ways or to induce Treg against the self-antigens. 15 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 In this table there are some examples of immune therapies applied to different kinds of pathologies; many of them are monoclonal antibodies that have been develop against different target such as cytokines or some specific molecules expressed by the autoreactive T or B cells. In some cases, there is the administration of cytokines themselves, like for the multiple sclerosis: it has been demonstrated that the injection of interferon β decrease the proliferation of autoreactive T cells and meliorate the symptoms. The anti-TNF, IL10, TGF-β decrease the pro-inflammatory cytokines but some molecules, expressed by the autoreactive T cells, increase the production of ADCC (antibody dependent cellular cytotoxicity) or CDC (complement dependent cytotoxicity) against the autoreactive T cells. These antibodies, for example anti-rituximab and CD20, can directly induce the death of the T or B cells through the NK or the complement. In this case is not self-antigen specific but is more specific than immunosuppressive drugs, that switch off all the inflammatory response, because in this case only the autoreactive one is blocked. 16 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 To induce the switching off of specific T or B autoreactive clone, different strategies has already been proven in the experimental model: - By using some altered peptide epitopes, you can induce the activation of antigen specific Treg, so they will be activated by specific antigen, and will switch off any kind of activation in the presence of the antigen. - Use of T cell engineered to kill the reactive B cells or modify the specificity of Treg: by introducing the Treg (differentiated in vitro) with an engineered TCR, that is a specific for a self-antigen, you will give the Treg differentiated from his own T cells. - Use of dendritic cells that are rendered tolerogenic instead of immunogenic. To induce tolerance, it can be used different way to administrate the antigen: the insulin can be given orally in order to induce the tolerance against it to prevent the disease. These examples below are shown only for your curiosity: In this study they enrol patient with high susceptibility to develop type 1 diabetes, and oral insulin or a placebo is given to them. You can see that the ones treated with oral insulin develop less diabetes that the ones that were treated with placebo. 17 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 In other cases, other groups, used the modified peptide (citrullinated peptide) against the rheumatoid arthritis. Scientists develop a sort of vaccine using dendritic cells loaded with this citrullinated peptide, that is very tolerogenic. The vaccine is called Rheumavax and they prove that these modified dendritic cells, that were rendered tolerogenic in vitro just by inhibiting the NFkb (that is one of the master transcription factor that induce all the pro-inflammatory cytokine), were not producing pro-inflammatory cytokines, nor expressing the co-stimulatory molecule. They isolate, from the blood, dendritic cells and load them (cultured) with citrullinated peptide; then they refuse them again in the patient’s lymph node, draining the joint that was affected by the arthritis. These tolerogenic dendritic cells were rendering the T cells anergic. They prove that the reinjection of these tolerogenic dendritic cells was safe and that was inducing the regulatory T cells. This is a proof that sometime this kind of approach can be used to treat the autoimmune disease. Link of the article: https://pubmed.ncbi.nlm.nih.gov/26041704/ This is another study related to type 1 diabetes. They use some different peptide of insulin, specific in this case for HLA, associated with an increase of IL10 and in foxP3. 18 Francesco Grossi/Alessia Gecchele Lesson 20, Immunology, prof.Capello 16/12/2021 They applied this sort of vaccine, administering the placebo or the peptide from the insulin, with low or high frequency in different patient, 10 in each group. The one receiving in high frequency means that every week he was receiving this antigen; the low frequency means every 2 week and the last group is the placebo. They reported that in both cases, low or high frequency receiving peptide, the insulin remains stable, while in the placebo group the develop of diabetes was increasing. In the group that was receiving the peptide in high frequency, the IL10 production and the Treg concentration in circulation were also increasing. Another observation was that the one responding, among the ten- patient receiving the peptide and the vaccine, presents less T memory cells, especially CD8 cells, in circulation; this means that there was a less activation of T cells and less memory T cells against the beta cells epitopes (all the antigens that are known to be recognized by autoreactive T cells in diabetes). 19 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 Tumor immunology The tumor immunology represents the study of immune response in presence of tumor. The first very important question for immunologists is “are tumors immunogenic?” that means “are they presenting antigen that can be recognized by the immune system?” To answer to this question, some experiments have been performed using some tumor cells line (these cells can be split and expanded in vitro) isolated from the tumor mass of any kind of mice; thanks to these cells, by injecting them in normal mice, it was possible inducing the tumor growth. This was very important because it was no longer necessary to wait the appearance of a tumor to observe and study the immune system regarding the anti-tumor response. For example, it was possible to inject ten mice at one time to have some robust data from a statistic point of view. The second question that has to be answered is “are tumors able to induce a specific immune response (thanks to the ability of T and B cells to recognize something specific on tumor cells) and memory?” Tumor cells lines have been used as a sort of a vaccine: by treating in vitro tumor cells in order to inhibit their growth (with some chemical reagent such as mitomycin which inhibits the cells division, or by using radiation), you can have tumor cells still alive but without the ability to growth in vivo. So, these cells can be used as a vaccine. The same mouse, injected on one side with the vaccine, if it is again injected with the same tumor cell lines without adding the stop growing factors, it will not develop the tumor. This proves that the immune system of this mouse has been challenged by the vaccine and it is able to recognize the tumor cells, that were not growing but were expressing all the other features of living tumor cells, and so reject the second injection. The specificity was proven by using breast tumor cell lines as a vaccine for another group of mice and another tumor cell lines for the second injection: the tumor deriving from the second cell lines, will grow. This simple example is the prove that you can induce, using tumor cells, memory and a specific immune response and so the activation of the adaptive immune response. Tumors are transformation of normal cells in which growth controls has become deregulated. Tumors differ from their normal counterparts in antigenic composition and biological behavior and those antigens could represent the tumor ones that can be recognized by the immune system. To reach this conclusion, many scientists had to work a lot during the time: Paul Erlich (1909): he was studying the immune system but at that time the notions about it were few; he was the first one to have the idea of the “cancer immunosurveillance” and so the idea that the immune system was able to recognize the tumor cells, the transformed ones, and reject, fight, counteract them. Medawar (1950): he was performing some experiment about tumor transplantation; he thought that the rejection of the tumor in mice was not due to tumor specifical antigens, but 1 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 it was mostly due to the allograft recognition: at the time, syngeneic mice were not available; syngeneic mice are animals with the same MHC class I and II so they are identical. His thought was correct. M. Burnet and L. Thomas (1957): they coined the cancer immunosurveillance hypothesis. By using syngeneic mice, they assume that some normal cells can develop heritable genetic changes that are fought by the immune system. These strategies play out by the immune system, for them, were a sort of immunological factors or cell. Now we know that normal cells (same idea of Burnet and Thomas) are transformed by genetic alterations and those cells can became tumor ones. These changes reflect a difference on the protein pattern of the tumor cells compared to the normal ones and this difference is what is at the base of immunosurveillance. Unfortunately, now we know that during the recognition, some cells can survive and develop some resistance mechanism, other can become tolerant and so again survive. Immunosuppress environment was used to prove that the lack of the immune system was allowing the tumor grow in vivo. Stutman was one of the first to use a chemical carcinogen, such as methylcholanthrene, to study the development and the latency (the time needed for a tumor to grow starting from the onset -and so from the use of the chemical carcinogen-) of a mass tumor. He used some nude mice (without hairs and thymus, and so almost immunodeficient) and discover that in the same latency time they develop the tumor as the control mice. This was the prove, for Stutman, that the immune system didn’t have a relevant role. Rygaard and Polvsen repeated the experiment (this time lasting 7 months and not 3) using much more mice, treated with the same chemical reagent, and they reach the same result as the scientist before. The following experiments were performed by using the neonatal thymectomy (mice in which the thymus was removed at birth), the anti-lymphocytes serum (the serum is the liquid part of the blood and so anti-lymphocytes serum means that it’s coming from, for example, rabbit that have been injected with antigen from mice and so they produce antibodies against these cells) and athymic nude mice (that were not the perfect model to study the immune suppression but at the time it was not known). All these experiments confirmed the first hypothesis claiming that the immune system was not so important in tumor development. After some time, scientist discovered that nude mice are not completely immunocompromise: it is true that they don’t have the thymus but there is also an extra thymic maturation of the T cells; they generate in the bone marrow and then usually migrate into the thymus, but T cells can also come from the liver hematopoiesis at the birth and so thanks also to NK cells and B cells, they are not totally immunocompromise. The discovery of other lymphocytes population, such as γδ T cells and natural killer cells, was also very important to reach the final conclusion about cancer immunosurveillance. 2 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 The nude mice used by Stutman have been revealed to be very sensitive to the 3- methylcholanthrene: they develop in any case the tumor after the administration because this carcinogen is very aggressive. Scientists also found out that the period of observation of 3-7 months was too short to see spontaneous tumor formation due to the presence/absence of immune system. Even the epidemiology studies in the 1970-1980 were giving some controversial result because they revel that the uncommon cancers were increasing in immunodeficient/compromise people whereas the common ones (lungs, prostate, breast cancers) were not increasing; especially, among the uncommon cancer, most of them were deriving from viral infection. This was proving that indeed the immune system has a role, but it is involved mainly in recognized tumor associated with infectious agents. Few years later, M. Burnet and L. Thomas states respectively “If there was tumor immunity it would be invisible”, “The greatest trouble with idea of immunosurveillance is that it cannot be shown to exist in experimental animals”; it is clear that the cancer immunosurveillance was not easy to prove with the knowledge of the time. This theory was finally truly considered at the end of the seventy (1978): Only new studies allow to identify new immune populations, new murine models (such as KO and transgenic ones) and so to validate the cancer immunosurveillance concept. All these new discovers revealed that immunosurveillance represents only one of the concepts of the complex interaction between the immune system and cancer; this is way nowadays we talk about cancer immunoediting and not immunosurveillance. Experiments that prove the concept of cancer immunosurveillance: All these experiments were performed using RAG knock out mice: the recombinant-activating gene encodes for the RAG protein that is important in the recombination of the TCR and BCR domain; these means that these KO mice are not able to recombine the TCR and BCR and so they have no T and B cells because they don’t survive to the maturation process. They are immunocompromise mice because of the lack of the somatic lymphocyte antigen receptor rearrangement that leads to no peripheral αβ T cells, B cells, γδ T cells and NKT cells. It has been proved that in the RAG KO mice (red line), using the methylcholanthrene to induce the growth of the tumor, this grows faster comparing to the situation that occur in normal mice (blue line). These mice have only the innate cells and therefore are truly immunocompromise. 3 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 By looking at the spontaneous tumors we can see the WT mice (immunocompetent) and those knock out for RAG2 (RAG-/- mice, immunocompromise). Most of the immunocompetent mice (12 out of 33) develop some benign tumors. The majority of rag KO mice (31/32) develop spontaneous tumors and malignant ones. This was also proved for other types of immunocompromise mice: for example, the interferon γ receptor KO mice, STAT1 KO mice and the RkSk ones (STAT1-/- and RAG2-/-) develop more tumors comparing to the WT mice. These experiments also prove that some kinds of protein and factors of the immune system are crucial: for example, talking about the interferon, without the INFGR1, these mice are immunocompromise for what concern the tumor growth. The spontaneous tumors in the RkSk or in the RAG2 KO are more frequent than in the WT even for a longer period of observation. In the image it can be observed the very few percentages of WT mice with benign adenoma cells compared to those immunodeficient with benign and also malignant tumors. 4 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 Beside the INFγ another protein has been discovered to be crucial in the fighting of tumor cells: perforin (pfp). The knockout mice for perforin, indeed, of course with the expression of the tumor suppressor p53 compromise, survive less compared to the same mice with perforin. → the absence of perforin is impairing the functional of the immune system against tumors. Tumors that develop in immunocompetent and immunodeficient mice are different: even by using the same agent to induce the tumor, the results are different. Starting from an immunocompetent mouse, we take the tumor cell line and inject it into a naïve WT mouse; this is done also for the immunocompromise mouse. The tumor will grow, and a similar kinetics can be observed in the immunocompromised mouse; in the immunodeficient mice, half of them will have the tumor, half of them will reject the tumor. → these are different tumors: the ones that rose in the immunocompetent mice are called edited (because the presence of the immune system is making a difference), while in the immunodeficient case are called unedited. In the latter we can found some progressors cells which are less immunogenic and less antigenic, and the regressor: cells that are not edited by the immune system because this mouse is immunodeficient; for this reason, it was really immunogenic and so able to recognize and kill the immune system of the competent mice. This was the beginning of the immunosurveillance theory. 5 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 Since 2000, the immunosurveillance hypothesis has been developing in immunoediting hypothesis (also called the three Es hypothesis): the immune system not only prevents but also differently shapes the tumor. In the image below, the three pictures represent the three Es theory: the phases of the tumor growth have been identified in elimination, equilibrium, and escape phase. Tumor cells are different from the normal ones present in the environment and they start to grow because they lose their growth regulate factors; this induce an inflammatory response and many innate cells (NKT, NK γδ) are recruited into the site of interest. As the tumor grows, some of them will die and the residential phagocytes will destroy them; thanks to the interferon gamma secreted by the innate immune cells, the antigen from the dying tumor cells can be taken up and be presented to the T cells residing in the lymph node. Here the T cells can expand both CD4 and CD8 T cells which in turn will reach again the primary tumor and perform their function by killing the tumor cells. This is what’s happening against virus and bacteria in infected cells. Unfortunately, all these battles can be long lasting and even a decade may be required to reach the equilibrium phase. So, in this phase the immune system stresses the tumor cells which, on the other hand, are very smart and can fight it by developing tolerance, by secreting anti-inflammatory cytokines, …, in order to overcome the immunosurveillance and then become clinically relevant to the diagnostic test. 6 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 Among the hallmarks of tumor cells and cancer malignant cells, we have the ability to avoid the immunosurveillance and to deregulate the metabolism of both cancer and innate cells. In general, we can say that tumor cells are able to: Grow in a chronically inflamed microenvironment; Evade immune recognition; Suppress immune reactivity. The role of chronic inflammation The inflammation process is so important for cancer transformation because tumors grow in an inflamed microenvironment: this microenvironment is composed by many cells (beside the tumor ones that are only the 20% of the tumor) including the immune ones; these are recruited by the release of the chemokines, alarmins and danger signal, and react without the specific ability to kill only the tumor cells. They release all the pro- inflammatory cytokines and establish a sort of chronic inflammation; all these cytokines, metalloproteases, chemokines released by the macrophages, are helping the tumor progression. From the early stage of tumor maturation due to an inherited DNA damage caused by mutations that occur thanks to the presence of ROS and other inflammatory cells that lead to genetic instability, we have the promotion, the progression and then the invasion thanks to the re-modelling of the extra-cellular matrix. The final step is the metastatization and so the formation of some hypoxic environment that induce an increase in the angiogenesis and therefor favor the travelling of tumor cells. 7 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 In people affected by chronic inflammatory disorder has been observed an increase susceptibility or incidence of cancer; another prove of the relationship between the chronic inflammatory response and the onset of tumor is that there are a lot of innate immune cells in the infiltrate of the tumor and when these innate cells are evolving in suppressor cells, this is correlated with a poor clinical outcome. It has also been observed that when people are cured from a chronic inflammation, and they are taking anti-inflammatory drugs, the cancer incidence decreases. Evade immune recognition Tumor cells, as we previously said, are so smart that are able to avoid the immune recognition in different ways: - TAA negative clones: during the progression and especially during the equilibrium phase of the three Es, some of the cancer cells, that are in continuous progression and become more aggressive as the time pass, to avoid the immune recognition, stop the expression of some of the TAA (tumor associated antigen) that are the proteins so well recognized during the elimination phase. Without the up-regulation of these TAAs, tumors cells are less visible because they are ignored by our immune system. - MHC low clones: tumor cells can start to decrease the expression of the MHC class I/II and even the genes that are associated with the antigen presentation in the MHC I/II. - Clones resistant to the immune attack: some clones can become resistant to the perforin, to the INFγ signal by avoiding the expression of the receptor or the secondary mediator in the transduction of the INFγ pathways. Suppress immune reactivity Some tumor cells can start to express some inhibitory ligand or/& receptors, produce some anti- inflammatory cytokines, or recruit and induce the differentiation of suppressive cells. The inhibitory receptors are the same as the one that we have already seen in the tolerance: there is PDL1 on the side of the tumor cell or the B7 for the CTL4. 8 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 PDL1, is very important in the periphery and it is the main inhibitory receptors engaged by the tumor cells. In some cases, tumor cells, because of the presence of some oncogenes that affect the expression of PDL1, express the PDL1 directly. In other cases, they originally do not express, but because they are in presence of an immune system that at the beginning is starting to fight the tumor cells they release cytokines (especially INFγ that induces PDL1 in tumor cells, so it can be originally present or acquired by the tumor cells later on). There are a lot of other suppressive cells that can be recruited or can differentiate in the tumor microenvironment especially thanks to the release of some cytokines, like the G-CSF or CM-GSF that are recruiting or differentiating the myeloid cells in suppressive phenotype; these cells can be MDSC, which means Myeloid-derived suppressor cells, suppressive DC (dendritic cells suppressive) and TAM (tumor associated macrophages). These are the same cells that we have studied related to the inflammatory response against pathogen; in this case, from the same kind of cytokines released by the bone marrow and recruited on the tumor site, instead of being effectors (like against pathogens), they become suppressor thanks to the factors released by the tumor cells. Some of these factors, for example, induce the appearance and the differentiation of the NKT suppressor cells, MDSC, etc... Thanks to the release of some cytokines such as the TGFβ or suppressive enzyme like IDO (it decreases the amount of tryptophan that reaches the T cells), or through the activation of secretions in myeloid-derived suppressor cells (present in the tumor) and other factors, the T cells can be inhibited. To sum everything up, the MDSC are similar to those present in the infection but inside the tumor they differentiate in the suppressive ones. 9 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 The Myeloid-derived suppressive cells: There are two subsets of MDSC that are discriminated by the expression of a molecule called LY6G+/-; the LY6G+ (granulocytic) are the most suppressive ones while the negatives (myeloids) are mild suppressor. These cells are able to induce the nitrosylation (they are characterized by a very active arginase) of the TCR or of the chemokines receptor and so modify the phenotype of the T cells and suppress them. The modification of TCR means that it won’t be able to recognize its common antigen because, due to the presence of the nitrosyl group, it will be different; the same it’s for the chemokine’s receptors that are no longer able to respond to the chemokine’s attraction and so in some case the T cells will be trapped into the tumor area. The suppression can also happen through the secretion of ROS in the presence of NO. The TAMs: Macrophages usually differentiate, when they sense a particular signal, into M1 or M2; the M1s, that are usually those who fight the pathogens, in the tumor are inducing the immune response and inhibiting the tumor growth by producing ROS, TNFα (that kills the tumor cells) and IL12 (that differentiate in TH1 and then CD8 which kills the cancer cells). The M2s, that express the arginase and other kinds of markers, on the other hand, are promoting the cancer growth by helping the fibroblast and by releasing growth factor. 10 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 The NKT cells: They represent the third population of suppressive cells; there are two subsets of NKT cells: they both work against the pathogen but talking about a tumor, the type I will promote the tumor immunity mediated by CD4 and CD8+ T cells while the type II will inhibit the cytotoxic response. The others “bad guy” for the immunosurveillance of tumor are the T regulatory cells: they inhibit, even in the tumor, the activation of T cells. The Treg can affect the APC or the effector T cells and in both cases, they block the activation of the immune system against the tumor. The APC, due to the expression of the CTLA4-CD80/CD86 on the surface, are not able to co- stimulate the T cells during the recognition of the antigen; they also express other kinds of ligands (LAG3-MHCII) and in general, they express factors that induce the dendritic cells to be more tolerogenic because, in fact, they are immature DC. On the other side, Treg also affect effector T cells by secreting the anti-inflammatory cytokines, by limiting the concentration of IL2, by releasing and enriching the environment of cAMP and adenosine (which is very anti-inflammatory for the T cells), and by releasing the granzymes B that can directly kill the effector cells. The image above sums up what we have seen so far. → Tumor cells are antigenic and there is a spontaneous reaction performed by the immune system against the tumor cells (thanks to the tumor antigenicity) and a tumor become clinically relevant when it has acquired the capacity to escape the immunosurveillance. All these considerations have to be considered to develop effective immunotherapies. To develop an immunotherapy, the best target on the tumor cells has to be defined: this must be long lasting, and it has to induce an immune reaction that will develop memory and the ability to also recognize the metastasizing cells. We can also fight, after a surgical resection of the primary tumor, the recurrence of the tumor because if they express the same target I have chosen, they will be recognized by the memory cells. 11 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 Tumor antigens: There are many tumor antigens and they are recognized by T and B cells and not only by the innate immune system; they have been identified in different kinds of tumors in which they induce a specific response and can be exploitable for the vaccine. To identify them, in the ’50 a scientist exploits the ability of tumor cells to grow in vivo and so he took a tumor induced by a carcinogen, excites the tumor and use it to inject the same mouse that was developing the tumor: the tumor now was not growing. If you inject it in a syngenetic mice: tumor grows. If you inject it in a different normal mice, with also the lymphocytes (from the first original mouse developing the tumor) the rejection of the tumor will be observed. → The T cells are recognizing something on the tumor. Different kinds of approach have been used to identify the, what was called tumor specific antigens (TSA), then develop into tumor-associated antigens (TAA). At the beginning it was thought that tumor cells for sure express some proteins that are specific for the tumor cells and not present in the normal ones; what then became evident is that actually tumor cells express modify proteins or proteins express in an aberrant way, but not specific ones, compared to normal cells. Some proteins, like the ErbB or the CD20, are normal proteins that in tumor cells are aberrantly up- regulating, because the amplification of the genes, and so they become TAA that can be recognized by the immune system; some antigens (in normal cases) are ignored until their expression is amplified and so they become representative on the MHC pocket and can be recognized by the T cells. Some of the tumor antigens, like the cancer testis, MAGE, TRP-1, are expressed in the wrong cells: the first ones are usually express in the testis, but in the aberrant situation they become expressed in epithelial cells. Some other antigens like the embryonic antigens and the α-fetoproteins are normally expressed in embryos but become re-expressed in adult cells. → TAA are proteins mostly expressed also by the normal cells but de-regulated, expressed on the wrong cells or in the wrong time. Others TAAs are represented by some viral proteins: there are some viruses that, after the infection of the cells, favor their transformation. How these TAA have been identified? Genetic approach: it was based on the idea that T cells could recognize the tumor cells as different. For example, from the melanoma, tumor cells have been excited (thanks to the surgical resection) and trypsinized in lab, and in parallel, from the same patient, the blood has been taken to isolate the T cells. Then you can co-culture the tumor cells with the lymphocytes in order to expand those that are specific for some the antigen expressed by the tumor cells. How is the identification of the antigen carried out? From the bulk of tumor cells, you clone all the genes in different single plasmids used to transfect normal cells that are usually not killed by lymphocytes; then all this single clones is put with the lymphocytes expanded in vitro and the ability of them to kill the tumor cells need to be checked. If the lymphocytes are not killing, it means that they haven’t recognized nothing abnormal. When the killing activity is found, it means that the lymphocytes are recognizing a protein, expressed by a tumor cell, that can render the tumor visible to the immune system. By checking the colonies created from each gene, you can identify the TAA that is inducing the activation of the T cells. 12 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 Biochemical approach: it is similar to the genetic one. In this case, starting from tumor cells, expressing MHC class I, you can elute it by changing the pH using specific buffer, the peptide from the HLA pocket. By fractionating the elution, this peptide is used to pulse dendritic cells that are then put in co-culture with CTL to test their ability to kill the APC. When the killing activity is found, you can identify, thanks to the mass spectrometry, the full protein from which the peptide is deriving. SEREX (serological analysis by recombinant expression cloning): the mRNA isolated from the tumor cells, retrotranscribed in cDNA, is used to create a phage library containing each gene. This library is used to infect some bacteria in order to express the related protein; these proteins are then transfer on a nitrocellulose membrane which is put in contact with the serum of the patient to perform an immunoscreening: if some antibodies are present into the serum, they will bind the protein present on the membrane (and in this way you can identify the protein based on the phage that was used to transfect the bacteria). In the serum, some anti- human igG are added in order to create a bond between them and the antibodies that are bound to the protein. (we are not sure what she said) 13 Alessia Gecchele / Daniele Friolotto Lesson 21, prof. Paola Cappello 21/12/2021 SERPA (Serological proteome analysis): It is similar to SEREX but the starting material is not the mRNA but the tumor cells. Tumor cells are isolated, trypsinized, lysated to obtain a suspension of all the proteins present in the tumor cells; these are separated in two dimensions: o the first one is on a very tiny lane in which proteins are divided based on pH: each protein is moving, thanks to the electric field, and stopping when the pH is equal to the isoelectric point. → fragmentation of the protein based on their isoelectric point. Then this lane is put on a normal SDS-page gel, the electric field is applicated and o in the second dimension the fraction of proteins will be separated based on their molecular weight (there are many proteins with the same isoelectric point and so a second fragmentation is required). The 2D gel (that is transparent) is again transferred into a nitrocellulose membrane and the western blot is performed. After that, there is the incubation with the patient’s serum and a healthy serum. From the developing of the film, we will have signals from both but thanks to the comparison between the two serums, we can identify new proteins that are recognized by antibodies present only in the cancer patient; these proteins, after being stained with different buffers (i.e. blue comassi), can then undergo to mass spectrometry and sequencing to obtain their name. Genetic and Biochemical approach are quite old; SEREX and SERPA are more recent, and they allowed the identification of TAA thanks to the recognition of the immune system. 14 Daniele Friolotto / Matteo Magliano – Lezione n°22 – Immunology – Prof. Cappello – 11/01/2022 CANCER IMMUNOTHERAPY The professor started making a small resume about the last lesson before starting the lesson. We continue our discussion about cancer immunology, in particular the immunotherapy. We’ll discuss all the story that allows us to realize that there is a sort of surveillance from the immune system regarding the onset of tumor and the ability to recognize and also kill transformed cells immediately, before they grow and begin a palpable mass or a clinical diagnose mass. So we realize that is important to exploit the capability of the different comparts of the immune system to find cancer, and for this is also born cancer immunotherapy. Cancer immunotherapy has been defined in 2013, almost 10 years ago, from the Science Journal, because the very important knowledge that were actually reached in the global scenario of the immunosurveillance in the past year Cancer immunotherapy has been developed and designed with different kind of strategies: - The adaptive cell transfer: the transfer of cell between T and NK cells - Monoclonal antibodies to target tumor antigens or immune system components (enhancing the ability to fight cancer) - Vaccine - Toll like receptor agonist - ICD inducer: specific chemotherapy/radiotherapy drugs that induce tumor cell death, so a death in a context of good inflammation that induce the activation of the immune system and the activation of specific B and T cells. Strategies about immunotherapy against cancer can be divided in 2 big categories: Passive / adoptive and active immunotherapy. PASSIVE IMMUNOTHERAPY The first strategies involve the administration of monoclonal antibodies against the tumor cells. With this strategy we are not inducing an immune response, but we are giving passively some component of the immune system that will induce many effects.  Monoclonal antibodies can be effective in inducing the killing of tumor cells by themselves, or they can be conjugated with anti-cancer drugs (toxin or chemotherapy drugs), radioisotopes (they can be used to focalize the target radiotherapy).  They can be administered for themselves, so this can be called the primary line of the treatment, or they can be administered in combination with chemotherapy.  The toxicity of this antibody treatment is not due to the antibody protein itself but is much more related to the type of the antigen of the target recognized by the antibody. 1 Daniele Friolotto / Matteo Magliano – Lezione n°22 – Immunology – Prof. Cappello – 11/01/2022  It is important the choice of the antigen / vaccine and the target to develop monoclonal antibodies to fight cancer Here we can see the different potential of antibodies. The antibodies induce the activation of NK cells and in particular ADCC (antibody dependent cellular cytotoxicity) or the degranulation of granulocytes or the activation of phagocytosis by macrophages after the opsonization of the tumor cell. The therapeutic monoclonal antibodies can also activate the complement, so thanks to the complement mediated cytotoxicity (CMC) and MAC there will be the lysis. They can also trigger a specific molecule on the tumor cell, blocking the ligand, or inhibiting the receptor dimerization, like for example the trastuzumab that binds Her2, that is an overexpressed gene so on some kind of breast cancer cells and for this reason, for the high concentration of Her2 on the cell surface, it can bind the growth factor. But this monoclonal antibody (trastuzumab) can bind and inhibit these receptors. Or the trastuzumab can also induce the internalization, so the disappearance of the molecule on the cell surface and the possibility of the signal transduction. We can develop new monoclonal antibody thinking about the target and so thinking the potential effector mechanism that we want to induce. Based on the target we can think of conjugating with some bacterial toxins, killing of tumor cells requires antibody binding and internalization of the toxin. They can be conjugated with isotypes for imaging or therapy, internalization is not required for the killing of tumor cells. Even neighboring cells that have bound the antibody can be killed (bystander effect), that is also pretty difficult to avoid. We can also exploit the conjugation with cytotoxic drugs, so bring inside the cells (or inside the mass) the chemotherapy drug. Sometimes is very difficult for the drug to reach the inside of the mass because of the high vascularization, but if we use antibodies, they will be able to arrive to the tumor and diffuse a little bit better and focalize the action of chemotherapy. 2 Daniele Friolotto / Matteo Magliano – Lezione n°22 – Immunology – Prof. Cappello – 11/01/2022 We can also conjugate the monoclonal antibodies with enzyme that activate pro-drugs. The following table shows the type of monoclonal antibodies, the target (most of them are checkpoint inhibitors), the years of approval and the cancers they have been approved on. Another big passive immunotherapy is the adoptive therapy, it is considered passive because we give to the patient some killer cells isolated from the blood of the patient, that have been expanded in vivo and reinfused in patient (again we can see it doesn’t give the patient instantly an inflammation). LAK cells (Lymphokine activated Killer) were generated in vitro in presence of IL-2, otherwise today we use the CIK cells (cytokine induced killer) that are expanded in vitro from the peripheral blood in the presence of IL-2, IL-15, INF-𝛾. In both cases they develop and expand killer cells that are very similar to CTL and NK cells. In this case more than 200 patient that receive LAK in combination of IL-2 for some days (just to maintain LAK cells) 5% of patient have displayed complete response, so the cells managed to completely reject 3 Daniele Friolotto / Matteo Magliano – Lezione n°22 – Immunology – Prof. Cappello – 11/01/2022 the tumor. 4% a partial response, where the tumor size is significantly reduced and also the metastasis disappears (so the 9% had a response). Other 4 % had an iatrogenic death. Adoptive cell transfer (ACT) Another population involved in this kind of therapy, the adoptive cell transfer (APC), is the T population There are three strategies for ACT therapies: 1) Tumor infiltrating lymphocytes therapy (TIL): lymphocytes obtained by biopsies, in lab is just minced and you can isolate the CD45, so all the leukocytes from the tumor cells. This lymphocytes are then expanded in vitro with IL-2 and are reinfused into the patient. They must also be treated with drugs or radiation to deplete their endogenous immune cells because otherwise we will have too many cells dangerous for the patient, which need allow the newly infused T cells to gain hold and fill the body. This kind of treatment is successful against melanoma. We collect the TILs from the tumor (is almost always infectable). Immune depletion with chemotherapy or radiation allows introduced T cells to take hold and multiply. After that they are reinfused into the patient and they will attack the tumor. They are similar to the LAK (the LAK are the peripheral blood lymphocytes) activated with IL-2. In this case they are maybe inside the tumor because they are already fighting the tumor. In vivo and in vitro they were more efficient than LAK. They were reviewed by Steve Rosenberg, that started the Steve Rosenberg’s saga, because for many years he improved the strategy and obtained very good result: For example he obtained 36% of Partial Response (so a partial regression) of tumor metastasis in patient with melanoma. 4 Daniele Friolotto / Matteo Magliano – Lezione n°22 – Immunology – Prof. Cappello – 11/01/2022 The strategy involves the isolation of the TILs from the tumor, then we have the culture of the TIL with the antigen (expressed/ aberrantly expressed / present by the tumor). We make an ELISA with INF-γ to select the cells that are really activated by the antigen. Then we take all the specific clones that respond to the tumor antigen and expand them, after that we will reinfuse these cells inside the patient. 5 Daniele Friolotto / Matteo Magliano – Lezione n°22 – Immunology – Prof. Cappello – 11/01/2022 In this study we can see 13 patients, half of them displaying a PR (partial response) and 3 of them show the Vitiligo. This means that these TILs specific for the antigens can also recognize the sa