MHC Molecules and Antigen Presentation Lecture PDF

MHC molecules and antigen presentation (lecture) Lilla Buzgó 1  MHC molecules and antigens Immune system Defence  Response reaction The phases of th...

MHC molecules and antigen presentation (lecture) Lilla Buzgó 1  MHC molecules and antigens Immune system Defence  Response reaction The phases of the response: 1. Recognition of the pathogen/danger signal 2. Alerting and mobilization of other components of the immune system 3. Elimination of the pathogen as soon as possible The most important phases of the response:  detection of pathogen or danger signal,  alarming and mobilization of other elements of immune system (including both the innate and the adaptive components),  elimination of the pathogen as soon as possible. The main function of the immune system is protection of the body against various pathogens, different biological toxins and even against altered, dangerous, abnormal cells inside the body. The two chief and clearly distinct functions of the immune system are recognition of the 2 dangerous and/or pathogenic structures followed by the elimination/neutralization of these structures. 2  MHC molecules and antigens Recognition Self Non-self (foreign material, pathogen etc.) Is there any abnormality? Dangerous? (e.g. tumor) Continuous monitoring requires continuous presentation! MHC T antigen- antigen lymphocyte presenting receptors cells B antigens lymphocyte epitope It is important to note, that not only pathogens can be considered as antigens. Antigen recognition could involve the recognition of the self-derived materials, but normally these don’t provoke a destructive immune response. The immune system is normally tolerant to self-antigens. According to the response following antigen recognition, we can classify immune responses into immunogenic and tolerogenic immune responses, and antigens into immunogenic and tolerogenic antigens (as discussed later). 3  Antigens Detection Antigens What is the antigen??? An entity that is specifically recognised by certain cells of the immune system (B and T lymphocytes) via antigen receptors and triggers a specific immune response (tolerant or eliminative) There can be a very large variety of antigenic peptides (10 15-17)  highly specific antigen receptors (the immune system can generate an astonishing variety of these receptors - 10 7-9 ) With such specific recognition, the immune system not only recognises pathogens, but also the body’s self structures that do not normally trigger an immune response The concept of antigen is easily understandable, yet antigen is one of the most ill-defined terms in immunology. As a general definition, the antigen is an entity recognized specifically by the B- and T-lymphocytes. The focus of this definition is on “specificity”. 4 This presumes the presence of highly specific receptors that can bind to a given material or structure, but fail to recognize others. These extreme specificities have been demonstrated by experiments in which some small chemical modifications of an antigen can render an antigen unrecognizable by the receptor which had been recognized in its original form. Or it may work the other way around; after chemical modification the altered antigen can be recognized with high specificity by another receptor which, 6 before the modification, 4 was unresponsive to it. Though in most cases small changes do not cause such a drastic effect in antigen recognition: modifications usually result in small changes in the binding affinity of the receptor to its specific antigen. The immune system can produce a vast variety of highly specific antigen receptors (in the order of billions). This receptor diversity is achieved by an elegant and sophisticated molecular genetic mechanism. An individual cell produces only one type of antigen-specific receptor, so one lymphocyte is specific to only one type of 4 antigen. Based on these we can create a simple but seemingly circular definition: The antigen is an entity recognized by the antigen receptors. It is important to note, that not only pathogens can be considered as antigens. Antigen recognition could involve the recognition of the self-derived materials, but normally these don’t provoke a destructive immune response. The immune system is normally tolerant to self-antigens. According to the response following antigen recognition, we can classify immune responses into immunogenic and tolerogenic 4 immune responses, and antigens into immunogenic and tolerogenic antigens (as discussed later). What terms should be used to describe immune recognition? Lipopolysaccharide, a bacterial cell wall component or a viral double stranded RNA should be considered as a PAMP, when it is recognized by pattern recognition receptors on various cell types. However, these structures are referred to as antigens when discussed from the point of view of lymphocytes that recognize them with their antigen-specific receptors, potentially capable of 4 recognizing minor chemical or structural differences in PAMPs. Generally the first recognition of the pathogens and other danger signals are mediated by PRRs, and the fine recognition and distinctions between self, non-self or modified self-molecules are mediated by lymphocytes with antigen receptors. Those molecules that can be recognized by antigen receptors and thus any material, that triggers a specific response from the immune system, either tolerance or a destructive immune response. 4  Antigens Cells of the immune system with specific antigen receptors: B lymphocytes T lymphocytes They carry a unique receptor capable of recognising only one type of antigen T Y Y B T Antigen One antigen recognition per cell B Antigen Y receptor BUT! T receptor B Billions of B and T cells  can recognise billions of different structures Proteins, carbohydrates, lipids, DNA, Recognised material Peptides derived from proteins (8-20 steroids, artifical compounds etc. amino acids) Natural form of antigen, may be tissue or Forms of recognition Presentation by antigen-presenting cells soluble (APC), via MHC molecule These cells carry a unique receptor that can only recognise one type of antigen. Thus, although each cell can recognise only one antigen, billions of B cells and billions of T cells can recognise billions of different structures. Most T lymphocytes recognize only pep tides, whereas B cells can recognize peptides, proteins, nucleic acids, polysaccharides, lipids, and small chemicals. As a result, T cell-mediated immune responses are usually induced by protein antigens (the natural source of foreign peptidesJ, whereas humoral immune responses are seen with protein 5 and nonprotein antigens. 5  Antigens Cells of the immune system with specific antigen receptors: B lymphocytes Naive T lymphocytes T Y B T Antigen Antigen B T Y receptor receptor Y B Antigen Antigen Differentiation and activation Differentiation Plasma cell cytotoxic effector T cell helper effector T cell (antibody production) B cells recognize the antigens by their cell surface antigen receptors. The antigen receptor of the B cell is a cell surface immunoglobulin. B cells express numerous membrane bound cell surface immunoglobulins, so one antigen can be bound by more 6 receptors at the same time. Following detection of antigens B cells get activated and differentiate into plasma cells. Plasma cells, the terminally differentiated form of B cells do not carry cell surface immunoglobulins, but they produce large amounts of these proteins in a soluble form which are commonly known as antibodies. Secreted immunoglobulins enter the surrounding tissue fluids and blood circulation. Secreted immunoglobulins or antibodies recognize the antigens of various pathogens and bind to them to 6 mark (called opsonization) or to inactivate them (called neutralization). Two main types of T cells are distinguished. The cytotoxic T cells specialized in the killing of infected or tumour cells and the helper T cells which play an important role in the facilitation and regulation of the immune responses. Helper T cells can enhance cytotoxic T cell responses, help macrophages to destroy the engulfed microbes and support antibody production of B cells. The two cell types can be easily distinguished based on their cell surface molecules. Cytotoxic T 6 cells express CD8 while helper T cells express CD4 cell surface receptors. Briefly, they are CD8+ (CD8-positive) or CD4+ (CD4- positive) cells. As has been repeatedly mentioned, please note that freshly generated so-called „naïve” T and B lymphocytes derived from the primary lymphoid organs are not fully functional! Further maturation/activation processes in the secondary 19 lymphoid organs or tissues are required for „naïve” lymphocytes to differentiate into functional effector cells. 6  Antigens Antigen recognition of naive T cells:  are mainly activated in secondary lymphoid organs  previously that have not met their specific antigen  their activation is mainly by dendritic cells (DC)  professional APCs  DC: in addition to MHC molecules, they express large amounts of cell surface molecules that provide efficient stimulation of naive T cells differentiation cytotoxic effector T sejt helper effector T sejt So far, little has been discussed about the antigen recognition of naïve T cells. Naïve T cells are activated in the secondary lymphoid organs. T cells that have not met their specific antigen can be most efficiently activated by dendritic cells. These professional antigen presenting cells in addition to the MHC molecules express those cell surface molecules, which provide effective stimulation for naïve T cells. Activated naïve T cells proliferate and differentiate into effector T cells, which later will be able to carry out the above mentioned functions of cytotoxic and helper effector T cells. Completing their development in the thymus both naïve CD4+ (“helper”) and naïve CD8+ T (“cytotoxic”) cells enter the circulation. Leaving the blood, they migrate into secondary lymphoid organs and screen the local antigen-repertoire on the surface of dendritic cells available at the time of their surveillance. A continuous recirculation of T cells between various secondary lymphoid organs is maintained until circulating T cells meet their specific antigen that induces their activation, followed by clonal expansion and differentiation into effector T cells. Clonal proliferation of T cells, like that of B cells rapidly generates a large population of cells (a T cell clone) with antigen specificity identical to that of their progenitor cell. Those T cells that fail to find their specific antigen are destined to die by apoptosis within a few weeks. 7 In most cases antigens for naïve T cells are transported into the lymph node by dendritic cells. Dendritic cells dispersed in tissues sense pathogens using their pattern recognition receptors. Upon recognition they engulf microbes and migrate into the regional lymph node. Meanwhile, processing of the pathogenderived antigen is completed and peptide fragments are presented on MHC. Thus, initial activation of T cells occurs in a special environment, the lymph node, where clonal expansion of antigen-specific T cells occurs. Those T cells that have gone through clonal expansion and differentiation exit the lymph nodes. Once in the periphery, they are able to deliver a proper response upon the second activation by the same antigen present at the site of infection. In the periphery, cytotoxic T cells (Tc) recognize their specific peptide fragment (antigen) presented by any nucleated cell expressing MHC I molecules. Notably, these antigen presenting cells recognized by Tc are usually infected or tumour cells. Helper T- 7 lymphocytes (Th cells) can only be activated by professional antigen presenting cells expressing cell surface MHC II molecules in peripheral tissues and also in peripheral lymphoid tissues. The T cells that have undergone proliferation (or clonal expansion) and differentiation are released to the periphery as effector cells, where they can respond appropriately to the cells presenting antigen. For the antigen-specific cytotoxic effector T lymphocytes clonal replicated in the lymph node, the antigen can be presented (as a peptide fragment) to almost any nucleated cell (that has become infected or transformed into a tumour cell) in the peripheral tissues. Only professional antigen-presenting cells that have MHC II molecules can present antigen to helper T effector cells: in peripheral tissues mainly macrophages, and in other peripheral lymphoid organs various B cells. 7  MHC molecules MHC = Major Histocompatibility Complex (HLA-Human Leukocyte Antigen)  The gene region with the highest polymorphism in the human population codominantly expressed  proteins with the highest diversity  MHC molecules with the same function but slightly different from each other  They are able to present peptide fragments from numerous types of antigens to T cells on the surface of the antigen-presenting cell (1 MHC molecule  1 peptide, BUT! one cell presents many different peptides)  Infection: microbial origin, Tumor: altered, "foreign" peptide sequences  T cells "control" peptides that bind to MHC molecules on the surface of the presenting cell and search for the specific MHC+peptide combination that activates them The MHC molecules (in human also called HLAs – Human Leukocyte Antigens), are encoded in the Major Histocompatibility Complex gene region. The MHC gene region encodes most of the proteins that are involved in the process of antigen presentation. This region contains the most polymorphic genes (with the highest number of allelic variations). As a result these genes encode proteins with the greatest diversity in the human population. Thus, most individuals of the population possess MHC molecules with slightly different structures but these molecules have identical function. The different MHC molecules encoded by different alleles can be recognised as “foreign” when some tissue or organ is transplanted into an other person, and the immune system will attack, reject the transpklant. This is why the name of this gene region refers to histocompatibility. On the cell surface, a vast number – even millions – of MHC molecules can be found simultaneously. One MHC molecule has affinity to various peptide sequences, but one MHC molecule binds only one peptide. All the MHC molecules on a cell surface together are able to present many different peptides from various different proteins at the same time to the T cells. Under normal circumstances only common self-protein derived peptides are presented by the help of MHC molecules. However, in the case of an infection microbial peptides can also appear on the cell surface presented by several MHC molecules among the normal self-peptide presenting ones. Tumour cell specific peptide fragments (from altered proteins) can be also presented among the normal self-peptides this way. By checking 8 tens of thousands of different peptides bound to MHC molecules on the surface of the antigen presenting cell, T cells are able to find a few specific MHC-peptide complexes, which activate them. MHC genes are codominantly expressed in each individual. In other words, for a given MHC gene, each individual expresses the alleles that are inherited from each of the two parents. For the individual, this maximizes the number of MHC molecules available to bind peptides for presentation to T cells. 8  MHC molecules Types of MHC: MHC I MHC II Genes ancoding MHC molecule: HLA- Genes encoding MHC II molecule: HLA-DP, A, HLA-B, HLA-C HLA-DQ, HLA-DR https://www.geeksforgeeks.org/major-histocompatibility-complex/ The structure of the MHC Class I is completely simple. It is a chain-like structure. The whole structure is made up of a chain. It is similar to the RNA structure. The chain has one end inside the membrane of the cell. The other end makes the structure of the MHC Class I. There are mainly three folds on the structure. They all are made up of a bend of the chain.There are two chains present. One is Alpha Chain & another is the Beta chain. Their chain is made up of the polypeptide molecule. There are three domains of alpha. They are Alpha1, Alpha2, Alpha3. There is only one domain of the Beta. The beta is completely detached from the cell membrane. The chain that is connected with the cell membrane, is made up of the domains of the Alpha.Alpha chains are non-covalently attached to the Beta. Also, there are transmembrane glycoproteins on the alpha domains. They act as the HLA gene. There are two di- sulfate bonds in the Alpha2 & Beta domain. They help to make a tight bond there. There are mainly two types of chains. There is an Alpha & Beta chain. But the structure is different from the structure of Class I. Here, two chains are anchored inside the cell membrane. Both chains have one side inside of the cell membrane. The other side makes the structure. There are two different changes. And those chains make different domains.The Alpha chain will create two domains. They are the Alpha1 & Alpha2. The Beta chain will also make two different domains. One is Beta1 & another is Beta2. All the chains will attach by non-covalent bonds. There 9 are di-sulfate bonds in the domain. Except for the Alpha2 domain, in every domain, there is the di-sulfate bond. This bond will help to hold the domain to its structure. The Alpha1 & Beta1 will jointly make the peptide bonding place. This is the place where the foreign substance will get attached. As a result, the signal will go to the T Helper cells to destroy the substances. -Each MHC molecule consists of an extracellular peptide-binding cleft, or groove, followed by immunoglobulin (lg}-like domains and transmembrane and cytoplasmic domains. As we shall see later, class I molecules are composed of one polypeptide chain encoded in the MHC and a second, non-MI-ICencoded chain, whereas class II molecules are made up of two MI-IC-encoded polypeptide chains. Despite this difference, the overall three-dimensional structures of class I and class II molecules are similar. -The polymorphic amino acid residues of MHC molecules are located in and adjacent to the peptidebinding cleft. This cleft is formed by the folding of the amino termini of the MI-IC-encoded proteins and is composed of paired a-helices resting on a floor made up of an eight-stranded ~-pleated sheet. The polymorphic residues, which are the amino acids that vary among different MHC alleles, are located in and around this cleft. This portion of the MHC molecule binds peptides for display to T cells, and the antigen receptors ofT cells interact with the displayed peptide and with the a-helices ofthe MHC molecules (see Fig. 5-1). Because of amino acid variability in this region, different MHC molecules bind and display different peptides and are recognized specifically by the antigen receptors of different T cells. We will return to a discussion of peptide binding by MHC molecules later in this chapter. -The nonpolymorphic Ig-like domains of MHC molecules contain binding sites for the T cell molecules CD4 and CD8. CD4 and CD8 are expressed on distinct subpopulations of mature T lymphocytes and participate, together with antigen receptors, in the recognition of antigen; that is, CD4 and CD8 are T cell "coreceptors" (see Chapter 7). CD4 binds selectively to class II MHC molecules. and CD8 binds to class I molecules. This is why CD4+ T cells recognize only peptides displayed by class II molecules, and CD8+T cells recognize pep tides presented by class I molecules. Most CD4+T cells function as helper cells. And most CD8+cells are CTLs. 9  MHC molecules Types ofMHC: Elsevier. Abbas et al.: Cellular and Molecular Immunology. 6th edition Class I molecules consist of two noncovalently linked polypeptide chains: an MHC- encoded 44-47 kD a chain (or heavy chain) and a non-MI-IC-encoded 12 kD subunit called ~2-microgIobuIin. Class II MHC molecules are composed of two noncovalently associated polypeptide chains, a 32 to 34 kD a chain and a 29 to 32 kD ~chain (Fig. 5-6). Unlike class 1 molecules, the genes encoding both chains of class II molecules are polymorphic. Az I. osztályú molekulák két, nem kovalens módon összekapcsolt polipeptidláncból állnak: egy MHC-kódolt 44-47 kD-s láncból (vagy nehézláncból) és egy nem MI-IC- kódolt 12 kD-s alegységből, az úgynevezett ~2-mikrogIobuIinból. A II. osztályú MHC- molekulák két, nem kovalens módon kapcsolódó polipeptidláncból állnak, egy 32-34 kD-s a láncból és egy 29-32 kD-s ~láncból (5-6. ábra). Az 1. osztályú molekuláktól eltérően a II. osztályú molekulák mindkét láncát kódoló gének polimorfak. 10  MHC molecules Binding of MHC molecule and the peptide:  They adopt a flexible conformation before the peptide binding  the peptide bond is accompanied by a conformational change that increases the stability of the complex  Bound peptide  contributes to stabilization of the conformation of the MHC+peptide complex  Peptide "capture" in the pocket  anchoring amino acids (residues)  different sequences  binding of peptides of different lengths  Noncovalent interaction  One MHC molecule  bind only one peptide at a time, but any peptide can bind Elsevier. Abbas et al.: Cellular and Molecular Immunology. 6th edition The binding of peptides to MHC molecules is a noncovalent interaction mediated by residues both in the peptides and in the clefts of the MHC molecules. T cell recognition of a peptide-MHC complex. This schematic illustration shows an MHC molecule binding and displaying a peptide and a T cell receptor recognizing two polymorphic residues of the MHC molecule and one residue of the peptide. Details of the interactions of peptides, MHC molecules, and T cell receptors are described in Chapters 5, 6, and 7. Each class I or class II MHC molecule has a single peptide-binding cleft that binds one peptide at a time, but each MHC molecule can bind many different peptides. Protein antigens are proteolytically cleaved in antigenpresenting cells to generate the pep tides that will be bound and displayed by MHC molecules (see Chapter 6). These peptides bind to the clefts of MHC molecules in an extended conformation. Once bound the peptides and their associated water molecules fill the clefts, making extensive contacts with the amino acid residues that form the p-strands of the floor and the a-helices of the walls of the cleft (Fig. 5-8). In most MHC molecules, the p-strands in the floor of the cleft contain "pockets." 11 The amino acid residues of a peptide may contain side chains that fit into these pockets and bind to complementary amino acids in the MHC molecule, often through hydrophobic interactions. Such residues of the peptide are called anchor residues because they contribute most of the favorable interactions of the binding (Le., they anchor the peptide in the cleft of the MHC molecule). The anchor residues of peptides may be located in the middle or at the ends of the peptide. Each MHC-binding peptide usually contains only one or two anchor residues, and this presumably allows greater variability in the other residues of the peptide. which are the residues that are recognized by specific T cells. Not all peptides use anchor residues to bind to MHC molecules, especially to class II molecules. Specific interactions of peptides with the a-helical sides of the MHC cleft also contribute to peptide binding by forming hydrogen bonds or charge interactions (salt bridges). Class I-binding peptides usually contain hydrophobic or basic amino acids at their carboxyl termini that also contribute to the interaction. Because many of the residues in and around the peptide-binding cleft of MHC molecules are polymorphic (Le.. they differ among various MHC alleles), different alleles favor the binding of different peptides. This is the structural basis of the function of MHC genes as "immune response genes"; only animals that express MHC alleles that can bind a particular peptide and display it to T cells can respond to that peptide. The antigen receptors of T cells recognize both the antigenic peptide and the MHC molecules, with the peptide being responsible for the fine specificity of antigen recognition and the MHC residues accounting for the MHC restriction of the T cells. A portion of the bound peptide is exposed from the open top of the cleft of the MHC molecule. and the amino acid side chains of this portion of the peptide are recognized by the antigen receptors of specific T cells. The same T cell receptor also interacts with polymorphic residues of the a-helices of the MHC molecule itself (see Fig. 5-1). Predictably, variations in either the peptide antigen or the peptide-binding cleft of the MHC molecule will alter presentation of that peptide or its recognition by T cells. In fact, one can enhance the immunogenicity of a peptide by incorporating into it a residue that strengthens its binding to commonly inherited MHC molecules in a population. 11  MHC molecules MHC I:  expressed on all cells with nuclei (except: red blood CD8+ cytotoxic cell) effector T sejt  linked to it peptides (8-10 amino acids long) formed T cell antigen receptor from proteins (intracellular or endogenous peptide origin) degraded in the cytoplasm of cell  abnormal MHC I proteins from tumour cells, proteins of bacterial or viral APC: antigen- origin  presentation of the internal environment of presenting cell the cell  for CD8+ cytotoxic effector T cells  intracellular antigen-presenting mechanism  presents any protein pathogen (no selection) Forrás: Gogolák P., Koncz G. : Bevezetés az Immunológiába - Avagy hogyan működik az immunrendszer Almost all cells of the body express MHC class I molecules (red blood cells can be mentioned as an exception). MHC I molecules bind peptides derived from proteins which are sliced in the cytosol. This is called as endogenous antigen presentation. Either the self-proteins of the cell or the proteins of intracellular bacteria, viruses or the abnormal proteins of the tumour cells can be presented this way to CD8+ cytotoxic T cells. With some exceptions (e.g. red blood cells) MHC I molecules are expressed on all human cells. They display mainly endogenous peptides on the cell surface for CD8+ cytotoxic effector T cells (Figure 9). Through the presented peptides, T cells can monitor what kinds of proteins are present inside the cells. Due to this process T cells could theoretically detect any intracellular pathogens, thus, antigen presentation by MHC I renders the intracellular space a subject for immunological monitoring or immunosurveillance. The antigen presentation process is Figure 9. Presentation of intracellular pathogens on MHC I MHC I molecules display peptide fragments which derive from proteins degraded in the cytoplasm. In case of an infection microbial peptides derived from intracellular pathogens are (also) bound to MHC I. Cytotoxic T cells recognize the MHC-I – peptide complex and may kill the infected antigen presenting cell. The antigen presentation process is not selective. It presents peptides 12 from any protein located in the cytosol (self/non-self), regardless whether it is derived from a pathogen or not. MHC I molecules can present peptides of the cell’s own proteins, but they can display peptides derived from intracellular bacteria or in case of a viral infection viral peptides can be also displayed. Based on the displayed peptides the T cells will decide whether there is any dangerous modification or infection of the antigen presenting cell. 12  MHC molecules MHC I antigen presentation pathway: Peptide  cytosol Proteasome: cleaves proteins into peptide fragments of the correct size (8-10 amino acid) Generated peptides TAP (transporter protein complex)  ER  binding to MHC I Peptide bound MHC I  through Golgi-apparatus appear on the cell surface „empty” MHC I molecule under normal circumstances can not found on cell surface! The MHC molecule becomes „closed”, unable to exchange the bound peptide https://www.geeksforgeeks.org/major-histocompatibility-complex/ It presents peptides from any protein located in the cytosol (self/non-self), regardless whether it is derived from a pathogen or not. MHC I molecules can present peptides of the cell’s own proteins, but they can display peptides derived from intracellular bacteria or in case of a viral infection viral peptides can be also displayed. Based on the displayed peptides the T cells will decide whether there is any dangerous modification or infection of the antigen presenting cell. MHC I bound peptides are generated in the cytoplasm (cytosol). The large multi-subunit housekeeping enzyme complex called the proteasome cleaves proteins into 13 peptide fragments of the correct size to allow complex formation with MHC I. The degradation of proteins in the cell is a natural process, as all kinds of cellular proteins are degraded by proteasomes. The generated peptides are delivered into the endoplasmic reticulum (ER) by a transporter protein complex. In the ER the freshly synthesized MHC molecules stabilised in peptide receptive conformation by chaperon proteins, so the transported peptides can bind to them. Usually 8-10 amino acids long peptides bind to the peptide-binding groove of MHC I molecules. Once an appropriate peptide has been bound to MHC I in the ER and the chaperons are dissociated, a conformational change occurs. The MHC molecule becomes „closed”, unable to exchange the bound peptide. In this peptide-bound state, the MHC I molecules can leave the endoplasmic reticulum and pass through the Golgi- apparatus, finally they appear on the cell surface. Under normal circumstances „empty” MHC I molecules, without a bound peptide, cannot be found on the cell surface. 13  MHC molecules If the MHC I molecule were not "closed" by peptide binding..... Peptides from the cell's extracellular environment could also be binding  healthy cells near the site of infection could become the targets  cytotoxic effector T cells kill them Other protective function: NK (natural killer cell)  lack of MHC I molecule activates  this can be useful for killing cells that try to hide from the adaptive immune system (virus-infected cells, tumour cells) The significance of this strict process is to prevent the binding of extracellular peptides to MHC I molecules on the cell surface, otherwise healthy cells near the site of infection could become the targets of cytotoxic effector cells. When the CD8+ cytotoxic T cells recognize peptides of foreign or harmful cellular proteins in complex with MHC I molecules, they kill the target cell to prevent further spreading of the infection or the development of a tumour. MHC I is expressed on the surface of all nucleated cells, so if any cell becomes infected, following antigen presentation, they become a target for cytotoxic effector T cells. In contrast to cytotoxic T cells, natural killer cells (NK cells) isn’t activated by MHC I bound peptides, but rather the lack of MHC I molecules, which unleash their activation. This mechanism can be effective in killing those cells, which lack MHC I molecules avoiding recognition by the adaptive immune system. For example 14 certain virus-infected cells express only a very limited number of MHC molecules, thus they become „invisible” to cytotoxic T cells. However NK cells detect the lack of MHC I and destroy the abnormal cells. 14  MHC molecules MHC II: CD4+ helper T cell  Present on professional antigen-presenting cell  professional APC: dendritic cells, macrophages, B lymphocytes T cell antigen receptor peptide  These cells endocytosis exogenous antigens (10-20 amino MHC II acids)  present them with MHC II to CD4+ helper T cells professional APC  Present antigens from extracellular space  presentation of the cell's external environment exogenous antigen Forrás: Gogolák P., Koncz G. : Bevezetés az Immunológiába - Avagy hogyan működik az immunrendszer MHC class II molecules show a much more restricted pattern of expression, being expressed mainly on the surface of the so-called professional antigen presenting cells. Using MHC II molecules, macrophages and dendritic cells present extracellular (exogenous) peptides derived from antigens engulfed from the extracellular space. Self-peptides of the body or foreign microbial peptides can be presented in complex with MHC II molecules, which can be recognized by CD4+ helper T cells. The MHC II molecules are expressed by the professional antigen presenting cells such as dendritic cells, macrophages and B cells. These cells are able to engulf extracellular antigens and to present them on MHC II molecules to CD4+ helper T cells. MHC II molecules (unlike MHC I) are specialized to display antigens derived mainly from exogenous origin (Figure 10). Following phagocytosis or endocytosis exogenous antigens get into 15 the endosome. Later the endosome fuses with lysosomes to become endolysosomes containing proteolytic enzymes. Peptides generated here by proteolysis –from exogenous proteins or from engulfed or parasitic microbes– can form a complex with MHC II molecules. Similar to MHC I, MHC II molecules are synthesized in the endoplasmic reticulum. However here they associate with a different set of chaperone proteins, most importantly with the so-called invariant chain (Ii). The main functions of the invariant chain is to form a complex with MHC II thus prevent binding of endogenous peptides into the peptide binding groove of MHC II and transport of the complex through the Golgi-apparatus in this ‘blocked’ state. In the endolysosome, proteolytic enzymes degrade the invariant chain, making the binding site available for peptides derived from extracellular antigens. Usually 10-20 amino acids long peptides bind to MHC II molecules, however, since its binding site is ”open at both ends”, longer, overhanging peptides are also able to fit. After peptide- binding, the MHC II-peptide complex appears on the cell surface to present antigens to CD4+ helper T cells. In return, activated helper T cells are able to influence the immunological functions of the antigen presenting cells in several ways:  Activated effector T-helper cells facilitate the activation and subsequent antibody production of the antigen presenting B cells, thus they support the humoral immune response. 15  In case of antigen presenting macrophages the effector T- helper cells further activate them, increasing their efficiency in killing phagocytosed bacteria “settled in” in their endosomes. 15  MHC molecules MHC II antigen presentation pathway: Exogenous Endocytosis Endosome (pH acidification)  Lysosome (proteolytic enzymes, cleave macromolecules) PEPTIDEs Endolysome MHC II molecules meet peptides (10-20 amino acids) from exogenous proteins in the endolysosome Cell surface MHC II molecule is generated in the ER  invariant chain (Ii) chaperone protein binds to it  prevents binding of endogenous peptides  Golgi apparatus  endolysosome  Ii is degraded  binding of exogenous peptides https://www.geeksforgeeks.org/major-histocompatibility-complex/ The professional antigen-presenting cells engulf the extracellular antigens and present them on MHC II molecules to CD4+ helper T cells. MHC II molecules (unlike MHC I) are specialized to display antigens derived mainly from exogenous origin (Figure 10).Following phagocytosis or endocytosis exogenous antigens get into the endosome. Later the endosome fuses with lysosomes to become endolysosomes containing proteolytic enzymes. Peptides generated here by proteolysis –from exogenous proteins or from engulfed or parasitic microbes– can form a complex with MHC II molecules. Similar to MHC I, MHC II molecules are synthesized in the endoplasmic reticulum. However here they associate with a different set of chaperone proteins, most importantly with the so-called invariant chain (Ii). The main functions of the invariant chain is to form a complex with MHC II thus prevent binding of endogenous peptides into the peptide binding groove of MHC II and transport of the complex through the Golgi-apparatus in this ‘blocked’ state. In the endolysosome, proteolytic enzymes degrade the invariant chain, making the binding site available for peptides derived from extracellular antigens. Usually 10-20 amino acids long peptides bind to MHC II molecules, however, since its binding site is ”open at both ends”, longer, overhanging peptides are also able to fit. After peptide-binding, the MHC II- peptide complex appears on the cell surface to present antigens to CD4+ helper T cells. 16 The resulting activation of CD4+ helper effector T cells can influence immunological functions associated with antigen-presenting cells: These activated helper effector T cells, by reflecting back to antigen-presenting B cells, can help activate them and promote antibody production, thus promoting an adaptive humoral immune response. In the case of antigen-presenting macrophages, helper effector T cells can further activate macrophages, which can then more effectively kill engulfed or endosome- inhabited bacteria. 16  Professional antigen-presenting cells Main function of professional APCs: Elsevier. Abbas et al.: Cellular and Molecular Immunology. 6th edition All the functions ofT lymphocytes depend on their interactions with other cells. For this reason, a great deal of effort has been devoted to defining how cell-associated antigens are displayed to T lymphocytes. Antigen-Presenting CellsAPCs are cell populations that are specialized to capture microbial and other antigens, display them to lympocytes, and provide signals that stimulate the proliferation and differentiation of the lymphocytes. By convention, APC usually refers to a cell that displays antigens to T lymphocytes. The major type of APC that is involved in initiating T cell responses is the 17 dendritic cell. Macrophages present antigens to T cells during cell-mediated immune responses, and B lymphocytes function as APCs for helper T cells during humoral immune responses. A specialized cell type called the follicular dendritic cell (FDC) displays antigens to B lymphocytes during particular phases of humoral immune responses. APCs link responses of the innate immune system to responses of the adaptive immune system, and therefore they may be considered as components of both systems. In addition to the introduction presented here, APC function will be described in more detail in Chapter 6. Dendritic Cells Dendritic cells play important roles in innate immunity to microbes and in antigen capture and the induction of T lymphocyte responses to protein antigens. Dendritic cells arise from bone marrow precursors, mostly of the monocyte lineage, and are found in many organs, including epithelial barrier tissues, where they arepoised to capture foreign antigens and transport these antigens to peripheral lymphoid organs. They have long cytoplasmic projections, which effectively increasestheir surface area, and they actively sample and internalize components of the extracellular tissue environment by pinocytosis and phagocytosis. In addition, dendritic cells express various surface receptors, such as Toll-like receptors (see Chapter 2), that recognize pathogen-associated molecular patterns and transduce activating signals into the cell. Once 17 activated, dendritic cells become mobile and migrate to regional lymphoid tissues, where they participate in presenting pep tides derived from internalized protein antigens to T lymphocytes. These activated dendritic cells also express molecules, called costimulators, which function in concert with antigen to stimulate T cells. They start to migrate into the draining lymph nodes through lymphatic vessels and they increase the expression of cell surface molecules essential for the activation of other immune cells. In the lymph nodes dendritic cells present the antigen in complex with MHC molecules for T cells (a more detailed description of the process will be provided in later sections). It is important to note that as the most effective professional antigen-presenting cells, dendritic cells are able to activate „naïve” T cells freshly released from the thymus. Mononuclear Phagocytes Mononuclear phagocytes function as APCs in T cell- mediated adaptive immune responses. We introduced mononuclear phagocytes (monocytes and macrophages) in the context of innate immune responses in Chapter 2. Macrophages containing ingested microbes display microbial antigens to differentiated effectorT cells. The effectorT cells then activate the macrophages to kill the microbes. This process is a major mechanism of cell- mediated immunity against intracellular microbes (see Chapter 13). Mononuclear phagocytes are also important 17 effector cells in both innate and adaptive immunity. Their effector functions in innate immunity are to phagocytose microbes and to produce cytokines that recruit and activate other inflammatory cells (see Chapter 2). Macrophages serve numerous roles in the effector phases of adaptive immune responses. As mentioned above, in cell-mediated immunity, antigen-stimulated T cells activate macrophages to destroy phagocytosedmicrobes. In humoral immunity, antibodies coat, or opsonize, microbes and promote the phagocytosis ofthe microbes through macrophage surface receptors for antibodies (see Chapter 14). As professional antigen-presenting cells (APC), macrophages participate in the induction of the adaptive immune response. In addition to engulfing and elimination of foreign antigens, dead cells or tissue debris of various sizes, macrophages also play an important role in the regeneration of damaged tissues, mainly by the secretion of growth factor. Follicular Dendritic Cells (FDC) FDCs are cells with membranous projections, which are present intermingled in specialized collections of activated B cells, called germinal centers, found in the lymphoid follicles of the lymph nodes, spleen, and mucosal lymphoid tissues. FDCs are not derived from precursors in the bone marrow and are unrelated to the dendritic cells that present antigens to T lymphocytes. FDCs trap antigens complexed to antibodies or complement products and display these 17 antigens on their surfaces for recognition by B lymphocytes. This is important for the selection of activated B lymphocytes whose antigen receptors bind the displayed antigens with high affinity(see Chapter 10). 17  MHC molecules MHC I MHC II CD8+ cytotoxic effector T cells CD4+ helper effector T cells Inducing the death of APCs they help and influences the APCs function, activation by the new cell surface molecules and cytokines E.g. by acting back on antigen-presenting B cells  activation, antibody production  adaptive humoral immune response antigen-presenting macrophages  further activation  more efficiently killing pathogens that are engulfed or colonised in the endosome After peptide-binding, the MHC II-peptide complex appears on the cell surface to present antigens to CD4+ helper T cells. In return, activated helper T cells are able to influence the immunological functions of the antigen presenting cells in several ways: Activated effector T- helper cells facilitate the activation and subsequent antibody production of the antigen presenting B cells, thus they support the humoral immune response. In case of antigen presenting macrophages the effector T- helper cells further activate them, increasing their efficiency in killing phagocytosed bacteria “settled in” in their endosomes. 18 Helper T cells or Th cells are not directly involved killing of pathogens, they rather coordinate the immune response by communicating with other immunocytes. Exogenous proteins processed to peptides by professional APCs and presented to CD4 co-receptor expressing Th cells via cell surface MHC II molecules. Antigen-specific activation of Th-cells via the TCR induces cytokine production and expression of novel cell surface molecules on the Tlymphocyte by which they coordinate the immune response. (Figure 19.) Cytotoxic T cells (killer cells, CTL, CD8+ T cell) recognize and kill “estranged”, virus-infected or tumour cells present in our body. Under physiological conditions our cells continuously synthesize and degrade cellular proteins. Peptides derived from these degraded proteins are transported to the cell surface and displayed in complex with MHC I molecules. The same mechanism operates for the presentation of viral- and tumour-associated proteins. Unlike Th cells, CTLs recognize antigen fragments in complex with MHC I molecules expressed by all nucleated cells, thus synthesis of foreign or mutant self proteins in any cells can be readily detected and eliminated by CTLs. (Figure 19.) Although the mechanism of recognition and activation of NK cells and CTLsare different, the mechanisms of killing the target cells are similar. Both celltypes make contact with the infected- or tumour-cell directly and releases 18 thecontent of their intracellular granules containing cytotoxic substances to thesite of cell-cell contact. Some of these cytotoxic substances like perforin molecules will form pores within the target cell membrane leading to disruption of the transmembrane ion balance which alone may be sufficient to cause the death of the target cell. Granzymes also released by these granules enter the target cell via perforin induced pores and trigger apoptosis. Additionally, effector CTL expresses molecules on the surface which induce apoptosis in the target cell. 18  MHC molecules B and T cells may respond to the same pathogen by amplifying each other’s response:  B cell  antigen binding in antigen receptor on cell  receptor mediated endocytosis  professional antigen presenting cells  presenting exogenous origin antigen by MHC II molecule  CD4+ helper effector T cell CD4+ helper effector T cell Cytocin production T cell antigen receptor peptide B cell Receptor mediated B cell antigen endocytosis receptor Exogenous origin Forrás: Gogolák P., Koncz G. : Bevezetés az antigen Immunológiába - Avagy hogyan működik az immunrendszer The peptides of the antigen inside the cell are displayed on the surface of the B cells by MHC II molecules, following the processing steps already described for the presentation of extracellular antigens. By this mechanism, the protein antigens recognised by B cells are also detected by T cells. Under these conditions, the B cell and the T cell have a good chance of recognising the same antigen, but the antigen receptor of the B cell and the T cell may not recognise the same part of the antigen. Antigen presentation through the MHC II molecule results in activation of helper T cells. The activated T cell may produce cytokines that promote B cell activation and 19 further differentiation. Cell surface receptors and their ligands that support or influence the activation of both cells may also interact during contact between the two cells. The two specific cell types, B and T lymphocytes, which recognise the same antigen, are thus able to support each other's function through a positive feedback loop (Figure 15). B and T cells can respond to the recognition of the same pathogen by amplifying each other. B cells, upon recognition of the antigen, can engulf it by receptor- mediated endocytosis. They can then present peptides from the protein antigen to helper T cells via MHC II molecules. Following antigen recognition, T cells produce cytokines that promote B cell activation and differentiation. Similar to dendritic cells and macrophages B cells are professional antigen presenting cells. The recognition of antigen by BCR induces not only activation, division and differentiation of the B cell, it also induces endocytosis of the bound antigen. The BCR-bound antigens can be efficiently internalized by B cells by the process of receptor-mediated endocytosis. Internalized antigens are processed and presented on the surface of B cells as peptide-MHC II complexes (as previously described in the concerning MHC II molecules). By this mechanism the protein antigens recognized by B cells can “be seen” by T cells as well. Under these 19 circumstances, chances are good that the B cell and the T cell will recognize the same antigen, but not necessarily the same part (epitope) of the antigen. Antigen presentation by MHC II leads to the activation of helper T cells. The activated T cell can produce cytokines that facilitate activation and differentiation of B cells. During cellular contact T and B cells mutually activate each other via cell surface receptors. Please note that in this process a positive feedback loop is created in which two lymphocytes (a T-and a B cell) with the same antigen specificity mutually support (as well as control) each other’s function (Figure 15.). 19  MHC molecules The expression of MHC molecules is enhanced by cytokines produced during the natural and adaptive immune response: IFN-α, IFN-β, IFN-γ, TNF (tumor necrosis factor), LT (lymphotoxin)  Enhancement of MHC I expression IFN-γ  the main cytokine involved in stimulating MHC II expression  may be produced by NK cells of the natural immune system or by antigen-activated T cells of the adaptive immune system Elsevier. Abbas et al.: Cellular and Molecular Immunology. 6th edition The expression of MHC molecules is increased by cytokines produced during both innate and adaptive immune responses (Fig. 5-10). On most cell types, the interferons IFN-a, IFN-~, and IFN-yincrease the level of expression of class I molecules, and TNF and LTcan have the same effect. (The properties and biologic activities of cytokines are discussed in Chapter 12.) The interferons are produced during the early innate immune response to many viruses (see Chapter 2), and TNF and LT are produced in response to many microbial infections. Thus, innate immune responses to microbes increase the expression ofthe MHC molecules that display microbial antigens to microbespecific T cells. This is one of the mechanisms bywhich innate immunity stimulates adaptive immuneresponses. The expression of class II molecules is also regulated by cytokines and other signals in different cells. IFN-y is the principal cytokine involved in stimulating expression of class II molecules in antigen-pre senting cells such as dendritic cells and macro phages (see Fig. 5-10). The IFN-y may be produced by NK cells during innate immune reactions, and by antigen-activated T cells during adaptive immune reactions. The ability of IFN-y to increase class II expression on antigen-presenting cells is an amplification mechanism in adaptive immunity. In dendritic cells, the expression of class II molecules also increases in response to signals from Toll-like receptors responding to microbial components, thus promoting the display of microbial 20 antigens (Chapter 6). B lymphocytes constitutively express class II molecules and can increase expression in response to antigen recognition and cytokines produced by helperT cells, thus enhancing antigen presentation to helper cells (Chapter 10). Vascular endothelial cells, like macrophages, increase class II expression in response to IFN-y; the significance of this phenomenon is unclear. Most nonimmune cell types express few, if any, class II MHC molecules unless exposed to high levels ofIFN-y. These cells are unlikely to present antigens to CD4+ T cells except in unusual circumstances. Some cells, such as neurons, never appear to express class II molecules. Human, but not mouse, T cells express class II molecules after activation; however, no cytokine has been identified in this response, and its functional significance is unknown. LT activates endothelial cells and neutrophils and is thus a mediator of the acute inflammatory response, providing a link between T cell activation and inflammation. These biologic effects ofLT are the same as those of TNF, consistent with their binding to the same receptors. However, because the quantity of LT synthesized by antigen- stimulated T cells is much less than the amounts of TNF made by LPS-stimulated mononuclear phagocytes, LTis not readily detected in the circulation. Therefore, LTis usually a locally acting cytokine and not a mediator of systemic injury. Lymphotoxin (LT) is a cytokine produced by T lymphocytes and other cells. It is approximately 30% homologous to macrophage-derived TNF and serves many of the same functions. A limfotoxin (LT) a T-limfociták és más sejtek által termelt citokin. Körülbelül 30%-ban homológ a makrofágok által termelt TNF-fel, és számos azonos funkciót lát el. TNF is the principal mediator of the acute inflammatory response to gram-negative bacteria and other infectious microbes and is responsible for many of the systemic complications of severe infections. The name of this cytokine derives from its original identification as a serum factor that caused necrosis of tumors. TNF is also called TNF- a to distinguish it from the closely related TNF-β, also called Iymphotoxin (LT). 20  Antigen presentation Antigen presentation is an all-time process in which antigen presenting cells can undergo at least as substantial changes as T cells , so the antigen presenting cell and the T cell affect each other. Elsevier. Abbas et al.: Cellular and Molecular Immunology. 6th edition Based on what we have learned so far, antigen presentation seems to affect the function of T cells only. In fact, during antigen presentation significant changes occur in the antigen presenting cells as well. The antigen presenting cell and the T cell mutually affect each other. Antigen processing is the conversion of native proteins into MHC-associated peptides. This process consists of the introduction of exogenous protein antigens into vesicles of APCs or the synthesis of antigens in the cytosol, the proteolytic degradation of these proteins into peptides, the binding of peptides to MHC molecules, and the display of the peptide-MHC complexes on the APC surface for recognition byT cells. Thus, both extracellular and intracellular proteins are sampled by these antigen- processing pathways, and peptides derived from both normal self proteins and foreign proteins are displayed by MHC molecules for surveillance by T lymphocytes. 21 For class II-associated antigen presentation, extracellular proteins are internalized into endosomes, where these proteins are proteolytically cleaved by enzymes that function at acidic pH. Newly synthesized class II MHC molecules associated with the Ii are transported from the ER to the endosomal vesicles. Here the Ii is proteolytically cleaved, and a small peptide remnant of the Iii called CLIp, is removed from the peptide-binding cleft of the MHC molecule by the DM molecules. The peptides that were generated from extracellular proteins then bind to the available cleft of the class II MHC molecule, and the trimeric complex (class II MHC exand pchains and peptide) moves to and is displayed on the surface of the cell.. For class I-associated antigen presentation, cytosolie proteins are proteolytically degraded in the proteasome, generating peptides with features that enable them to bind to class I molecules. These peptides are delivered from the cytoplasm to the ER by an ATP-dependent transporter called TAP. Newly synthesized class I MHC-pz- microglobulin dimers in the ER are attached to the TAP complex and receive peptides transported into the ER. Stable complexes of class I MHC molecules with bound peptides move out of the ER, through the Golgi complex, to the cell surface.. These pathways of MHC-restricted antigen presentation ensure that most of the body's cells are screened for the 21 possible presence of foreign antigens. The pathways also ensure that proteins from extracellular microbes preferentially generate peptides bound to class II MHC molecules for recognition by CD4+ helper T cells, which activate effector mechanisms that eliminate extracellular antigens. Conversely, proteins synthesized by intracellular (cytosolic) microbes generate peptides bound to class I MHC molecules for recognition by CD8+ CTLs, which function to eliminate cells harboring intracellular infections. The immunogenicity of foreign protein antigens depends on the ability of antigen-processing pathways to generate peptides from these proteins that bind to self MHC molecules. 21  Antigen presentetion Can it be inhibited? Yes….  Virus  co-evolved with their host  they have developed many strategies to evade immune responses  Several viral genes have been identified that encode proteins that modulate host immune responses. Some of these viral proteins inhibit the presentation of viral antigens to T cells.  Examples:  Pathogen adenoviruses  Herpes simplex  Human CMV  Kaposi sarcoma herpes virus  HIV As viruses have co-evolved with their hosts, they have developed many strategies to evade immune responses. Several viral genes have been identified that encode proteins that modulate host immune responses. Some of these viral proteins inhibit the presentation of viral antigens to T cells. The transcription of class I MHC genes is inhibited by the E1A protein of pathogenic strains of adenovirus. Herpes simplex viruses 1 and 2 produce a protein, called ICP-47, that binds to the peptide-binding site of the TAP transporter and prevents the transporter from capturing cytosolic peptides and transporting them into the endoplasmic reticulum for binding to class I molecules. The adenovirus E3 19-kD protein binds to and retains class I molecules in the endoplasmic reticulum, preventing these molecules from exiting with their peptide cargo. The human cytomegalovirus (CMV) US3 protein sequesters class I molecules in the endoplasmic reticulum, and murine CMV gp40 (40-kD glycoprotein) retains class I molecules in the cis-Golgi compartment. Human CMV produces two proteins, US2 and US11, which bind to class I molecules in the endoplasmic reticulum and actively carry, or dislocate," these molecules into the 22 cytosol, where they cannot be loaded with antigenic peptides and are degraded in the proteasome.. Kaposi sarcoma herpes virus (KSHV) K3 and K5 proteins induce rapid internalization of class I MHC molecules from the cell surface. Two proteins of HIV, Vpu and Nef, also inhibit class I expression in infected cells; Nef appears to do this by forcing internalization of class I molecules from the surface of infected cells, and Vpu destabilizes newly synthesized class I molecules. 22 MHC molekulák 23 Thanks for your attention! 24 Felhasznált irodalom: Gogolák P., Koncz G. (2015): Bevezetés az Immunológiába - Avagy hogyan működik az immunrendszer. Egyetemi jegyzet. Debreceni Egyetem. Abbas A. K., Lichtman A. H., Pillai S. (2007): Cellular and Molecular Immunology. International edition. 6th edition. Elsevier. ISBN: 978-0-8089-2358-9. Elsevier. Abbas et al.: Cellular and Molecular Immunology. 6th edition 25