مناعة كاملة مع تحيات مكتبة الخليج العربي PDF

The Syllabus of basic Immunology For third Medical Students 1. Overview of the immune system o History of immunology o Function of the immune system o Organs & microenvironments of immune system o Origin of immune cells o Arms of immunity...

The Syllabus of basic Immunology For third Medical Students 1. Overview of the immune system o History of immunology o Function of the immune system o Organs & microenvironments of immune system o Origin of immune cells o Arms of immunity o Components of innate & adaptive immunity o Essential differences and interaction between innate & adaptive immune response 2. Innate immunity o Characterization of innate immunity o Lines of defenses in innate immunity o Cells of innate immune responses o Receptors for innate immunity o Signaling through PRRs 3. Mechanisms of Innate immunity o Phagocytosis, Netosis, and Pyroptosis o Natural cytotoxicity by NK cells o Natural Cytostasis and interferons 4. Inflammatory response o Local and systemic inflammation o Regulation of innate immunity 5. Complement system o Complement Components o Major pathways of complement activation o Complement receptors o Functions of Complement components o Regulation of complement activity o Microbial complement evasion strategies o Complement deficiencies 6. MHC molecules and antigen presentation o Classes & Structure of MHC molecules o Role, expression patterns and inheritance of MHC molecules o Inheritance of MHC o Major Pathways of antigen processing & presentation o Presentation of Non-peptide antigens o Regulation of MHC expression o MHC deficiency & susceptibility of diseases 7. Ontogeny of Thymocyte o Thymocyte development & thymic selection o TCR complex structure o Mechanisms for generating of TCR diversity 8. T cell activation & differentiation o Signal for T cell activation 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 1  o Activation by superantigens o T helper subsets polarization & cross-regulation o Mechanism of T reg activity o Tc subsets and mechanism s of killing o Memory cell subsets 9. Ontogeny of B cell o B cell development in BM & periphery o Self-tolerance of autoreactive B cell o Basic structure of antibody &BCR complex o Mechanisms for generating of BCR diversity o B cell subsets 10. Activation & differentiation of B lymphocytes o TD versus TI activated B cells o Class switching, somatic hyper mutation & affinity maturation o Primary versus secondary antibody immune response o Affinity & avidity o Isotypes of antibody o Effector functions of antibody o Enzymatic digestion of antibody 11. Immunogenicity o Immunogenicity Versus Antigenicity o Factors affecting immunogencity o Epitops o Haptens & cross-reactivity o Antigen-antibody interactions Overview of immune system History of immunology Function of the immune system Organs & microenvironments of immune system Origin of immune cells Arms of immunity Components of innate & adaptive immune system Essential differences between innate & adaptive immune response Interaction of innate & adaptive immune system 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 2  Indeed, the field of immunology can be credited with the vaccine that eradicated smallpox, ability to transplant organs between humans, & drugs used today to treat asthma. In fact, it interleaves with many of the other body systems, including endocrine, nervous, & metabolic systems. Finally, it has become increasingly clear that elements of immunity play key roles in regulating homeostasis in the body for a healthy balance. Function of the immune system o Protection against infectious agents. The immune system (IS) evolved to protect multicellular organisms from pathogens. It defends the body against invaders as diverse as the tiny (~30 nm), intracellular virus that causes polio and as large as the giant parasitic kidney worm Dioctophyme renale, which can grow to over 100 cm in length and 10 mm in width. 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 3  o Immunologic surveillance to remove dead, mutated, altered or infected cells. o Immune system decides that a tissue belongs, then is tolerated and allowed to remain or rejected as in organ transplantation. The fully functional IS involves so many organs, cells, molecules & pathways in such an interconnected & sometimes circular process that it is often difficult to know where to start! Organs of immune system (lymphoid organs) o Primary lymphoid organs The primary or central lymphoid organs (LOs) are bone marrow & Thymus. o Secondary lymphoid organs The secondary or peripheral LOs include lymph nodes, spleen, & various mucus-associated lymphoid tissue (MALT) such as Tonsils, gut- associated lymphoid tissue (GALT). o Tertiary lymphoid tissue The tertiary lymphoid tissues which normally contain fewer lymphoid cells than secondary LOs, most of these are cutaneous-ALT 4 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 4  ‫‪5‬‬ ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ ‫‪5‬‬ Arms of immunity Vertebrates have two arms: Innate immunity Adaptive immunity Humoral immune response Cell-mediated immune response 6 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 6 Components of the innate and adaptive immune immunity Innate immunity Adaptive immunity Anatomic & Skin, mucosa, normal flora, Lymph Nodes, physiologic barriers temp., PH, Spleen, MALT Monocytes/macrophages B cells/plasma cells Cellular components Neutrophils T cells Eosinophils Basophils Mast cells Natural killer cells Secreted Antimicrobial peptides Antibody components Complement Cytokines Cytokines Lysozyme Acute phase proteins Interferons Essential differences between the innate and adaptive immunity Characteristics Innate Adaptive Time of response Provides a rapid response The response takes time to develop, Specificity of receptors For Pathogen associated For specific antigens of molecular patterns (PAMPs) microbial & non microbial agents Diversity Limited High Memory No, so the response does not Yes, so subsequent improve with repeated exposure infections with the same antigen are therefore dealt with more quickly 7 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 7 This very basic scheme shows the sequence of events that occurs during an immune response, highlighting interactions between innate and adaptive immunity. In this example, bacteria breach the mucosal lining of the throat, a skin or mucous barrier, where it is recognized and engulfed by a local phagocytic cell (step 1). As part of the innate immune system, the phagocytic cell releases cytokines and chemokines that attract other white blood cells to the site of infection, initiating inflammation (step 2b). That phagocytic cell may then travel to a local lymph node, the tissue where antigen and lymphocytes meet, carrying bacterial antigens to B and T lymphocytes (step 2a). Those lymphocytes with 8 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 8 receptors that are specific for the antigen are selected, activated, and begin the adaptive immune response by proliferating (step 3). Activated TH cells help to activate B cells, and clonal expansion of both types of lymphocyte occurs in the lymph node (step 4). This results in many T and B cells specific for the antigen, with the latter releasing antibodies that can attach to the intruder and direct its destruction (step 5a). The adaptive response leaves behind memory T and B cells available for a future, secondary encounter with this antigen (step 5b). Dr. Dekra El-Aghbary Associate Professor of Immunology 9 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 9 Innate immunity Characterization of innate immunity Lines of defenses in innate immunity Cells of Innate immunity Receptors for Innate immunity Characterization of innate immunity  Early protection from infection  The innate immune defenses are all present at birth;  Activated very soon after pathogen exposure or opportunistic microbe reactivation  They have a very limited diversity for antigen, & attack microbes with the same basic vigor no matter how many times they have seen the same pathogen. Lines of defenses in innate immunity First line of defense Anatomical & physiologic barrier Skin & epithelial layers insulate the interior of the body from outside pathogens Epithelial layers of mucosal tracts & secretory glands produce a variety of protective substances, including acidic pH, enzymes, binding proteins, antimicrobial proteins & peptides. 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 10  Due to its size & critical roles as barriers, many call the skin is the most imp. immunological organ in the body. 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 11 Second line of defense (Cellular innate response) 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 12  ‫‪4‬‬ ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ ‫‪13‬‬ Monocytes/MQs: Monocytes circulate in blood, become MQs in tissues, characterized by: Phagocytosis Prolonged defense. Produce cytokines that initiate & regulate inflammation. Professional antigen presenting cell (APC), Clear dead tissue & Initiate tissue repair Macrophages will develop along one of 2 different pathways 1. M1 (Classical) Induced by innate immunity (TLRs, IFN-γ) Phagocytosis & initiate inflammatory response 2. M2 (Alternative) Induced by IL-4, IL-13 Tissue repair & control of inflammation 5 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 14  Receptors for Innate immunity Antigen presenting cells (APCs) recognize conserved microbial components, pathogen-associated Molecular patterns (PAMPs) such as bacterial LPS & peptidoglycans, as well as viral ssRNA & dsRNA. These PAMPs serve as ligands for a broad array of protein families referred to as pattern recognition receptors (PRRs). PAMPs can be expressed by microbes whether or not they are pathogenic, hence they are sometimes referred to as microbe-associated molecular patterns (MAMPs). PRRs recognize MAMPs that are common to whole classes of microbes. Some PRRs are also capable of recognizing molecules released by damaged, dying, or dead host cells, referred to as damage-associated molecular patterns (DAMPs). Until now, there are Five families of PRRs; Toll-like receptors (TLRs), C -type lectin receptors (CLRs), NOD-like receptors (NLRs), RIG-1-like receptors (RLRs), & AIM-2-like receptors (ALRs). A single innate immune cell may express multiple types of innate receptors, & many innate cells can express same PRRs. This means that every antigen that is capable of binding to a particular innate receptor can be bound immediately by many cells. Receptor name Cellular Location (s) Pathogen target Downstream effect Toll-like Extracellular Microbial CHO, Production of Receptor (Plasma mem.) lipoproteins, fungal mannans, antimicrobials, (TLRs) Intracellular bacterial flagellin, viral RNA, antivirals, & cytokines; TLR-1 to 13 (endosomes & self-components of damaged Inflammation Lysosomes) tissues, etc. C-type lectin Extracellular CHO components of fungi, Phagocytosis, Receptor (Plasma mem.) mycobacteria, viruses, production of (CLR) parasites, & some Allergens antimicrobials & cytokines; NOD-like Intracellular Fragments of intracellular or Production of Inflammation Receptors (cytosol) extracellular bacteria cell wall antimicrobials & (NLRs) peptidoglycans cytokines; Inflammation RIG-like Intracellular Viral RNA IFN production & Receptors (cytosol) cytokines (RLR) AIM-like Intracellular Viral and bacterial DNAs Production of IFNs & Receptor (cytosol & Nucleus) cytokines (ALR) 6 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 15  In addition, many different cell types express PRRs, including keratinocytes, epithelial cells, & even B and T lymphocytes. After PRRs recognize a specific of ligands as PAMP & DAMP & they trigger signaling pathways which activate a variety of genes encoding proteins that contribute to innate & inflammatory responses. After PRRs recognize a specific of ligands as PAMP & DAMP & they trigger signaling pathways which activate a variety of genes encoding proteins that contribute to innate & inflammatory responses. These genes, including those for Antimicrobial peptides, Type 1 interferons (IFNs), Cytokines & chemokines & Enzymes that help to generate antimicrobial & inflammatory responses. Cytokines Cytokines function as the hormones of immune system, produced in response to stimuli & acting on a variety of cellular targets. Major proinflammatory cytokines induced by PRR activation during innate immune responses are IL- 1, TNF-α, and IL-6. They act locally on blood vessels to increase vascular permeability & also on other cells, including lymphocytes, to recruit & activate them at sites of infection. They also have systemic effects, including inducing fever & feeding back on BM hematopoiesis to enhance production of neut., & other myeloid cells that will contribute to pathogen clearance. 7 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 16 Chemokines Chemokines are small protein chemoattractants recruit cells into, within, & out of tissues. Some chemokines are responsible for homeostatic migration of white blood cells throughout body. Other chemokines, produced in response to PRR activation, have key roles in the early stages of immune & inflammatory responses in that they attract cells such as IL-8 and that they contribute both to clearing infection or damage & to amplifying response. Enzymes Two enzymes produced in response to PRR-activated signaling pathways, inducible nitric oxide synthase (iNOS) & cyclooxygenase-2 (COX2) have key roles in generation of antimicrobial & proinflammatory mediators. iNOS enzyme catalyzes an important step in formation of nitric oxide, which kills phagocytosed microbes. COX2, whose produced by monocytes, MQs, neut., & mast cells, is key to converting lipid intermediate arachidonic acid to prostaglandins, potent proinflammatory mediators. Dr. Dekra El-Aghbary Associate Professor of Immunology 8 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 17 Mechanisms of cellular innate defenses Phagocytosis, Netosis and Pyroptosis Natural cytotoxicity by NK cells Natural cytostasis and Interferons Phagocytosis Phagocytosis is the ability of specialized cells, phagocytes (mainly neut., & MQs), to internalize pathogens & destroy them. Phagocytic cells ingest & digest particulate debris, such as microorganisms, host cellular debris, & activated clotting factors. In addition to the binding of PRRs to PAMP on the pathogen, opsnization also are enhanced the phagocytosis. 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 18  Neutrophils release granule contents into extracellular milieu during phagocytosis & inflammation in which the neutrophils die, forming what is known as pus. 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 19  Incomplete phagocytosis is the inability of phagocytes (mainly MQs) to destroy internalized pathogens when these pathogens persist and can spread throughout the body. After that, NETs inactivate pathogens by means of antimicrobial proteins, NETs may be cleaned away by MQs that engulf & degrade them. ROS get released into tissues and may damage tissues. Recent investigations suggest that NETs may play an essential role in both defense against infections & the pathogenesis of thrombotic disorders. Pyroptosis Pyroptosis is a highly inflammatory form of lytic programmed cell death that occurs most frequently upon infection with intracellular pathogens and is likely to form part of the antimicrobial response response. The Greek pyro refers to fire and ptosis means falling. The term pyroptosis may be understood as "fiery falling", which describes the bursting of pro- inflammatory chemical signals (IL-1 & IL-8) from the dying cell. 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 20  NLRs are a large family of cytosolic PRRs activated by intracellular PAMPs, DAMPs, & other harmful substances. The binding of NLRs with intracellular microbial components such as cell wall fragments lead initiate signaling pathways. Some NLRs assemble into inflammasomes, large protein complexes that with caspases cleave & activate the proinflammatory cytokines IL-1β and IL-18 & induce death of activated MQs through pyroptosis. In a cell that undergoes pyroptosis, chromatin condensed, however, the nucleus remains intact and gasdermin pores are formed on the plasma membrane, resulting in water influx. 4 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 21 Mechanisms of Killing by NK cells Natural killer cells Natural killer (NK, large granular lymphocytes) cells were discovered in the early 1970s as a population of cells that could kill some tumor cells without previous exposure some tumor cells without previous exposure. NK cells develop from common lymphoid progenitor cells in BM. They belong to innate lymphoid cells (ILC) that lack antigen-specific B- and T-cell receptors & play important roles in innate immune responses. Function of NK cells NK cells are an important part of the early innate response to viral infections as well as to malignancy & other indicators of danger. These early innate responses control infection for days to week it takes for adaptive response to be generated. Activated NK cells secrete proinflammatory cytokines such as TNF-α, & IFN- γ a potent MQ activator that also helps to activate & shape adaptive response. 5 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 22 Receptors of NK cells NK cells do not express antigen-specific receptors (TCR) or CD3. Their recognition of targets is not (Major histocompatibility protein) MHC- restricted. They do express two types of receptors, which deliver either activating or inhibitory signals to target cells. NK-cell inhibitory receptors (KIRs). NK-cell activating receptors (KARs). NK cell surface receptors responds to a balance of activating & inhibiting signals delivered by self-cell Many transforming viruses can induce-down regulation of MHC expression which lead to identify by NK cells & eliminate them. NK cell stimulated by cytokine as type I IFNs & IL-2, IL-12, & IL18 produced by DCs, MQs, neut., & mast cells. The markers used clinically to enumerate NK cells include CD16 (FcRγ) & some PRRs such as TLRs, RIG-I & cell adhesion molecules (CAM). 6 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 23 Mechanism of killing by NK cells 1. Fas/FasL-mediated cytolysis The interaction of FasL with Fas on a target cell triggers apoptosis by Fas pathways 2. Antibody-dependent cell mediated cytotoxicity NK cells bind to antibody on target cells by FcγR on their surfaces. Activation of NK cells leads to a release of perforin piercing target cell membrane, running granzyme through pore inside cell, & initiate caspase cascade, inducing apoptosis in target cell. 7 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 24 Interferons and Natural Cytostasis IFNs is a family of cytokines, including Type I IFNs (IFN-α & IFN-β) & Type II IFN (IFNγ) They have potent antiviral actions by inducing certain enzymes inside virus- infected cell & certain tumor cell. IFN-inducible enzymes control all steps of viral replication, as a result, natural cytostasis takes place. Type I interferons Type I interferons (IFN-α & IFN-β), are secreted by many cell types including lymphocytes (NK, B, & T cells), MQs, DCs, fibroblasts, virus-infected cells, endothelial cells, osteoblasts, & others. They are secreted under influence of some inducers such as viral envelope glycoproteins, CpG DNA, dsRNA, & other PAMPs tumor- associated molecular patterns (TAMPs). Both IFN-α and IFN-β bind use CD118 molecule as receptor & induce similar biologic responses. 8 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 25 Activities of Type I IFNs: Induces an antiviral state in most nucleated cells; Activates NK cells, Increase expression of class I MHC molecules, & IFNα acts as a pyrogenic & painful factor by affecting thermosensitive neurons in hypothalamus that causes fever & pain. 9 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 26 Natural cytostasis Main effector pathways of IFN-mediated antiviral response are as follows: 1. Protein kinase R (PKR) inhibits translation of viral proteins. 2. 2′,5′-Oligoadenylate-synthetase (OAS) mediates RNA degradation. 3. Mx protein GTPases inhibit RNA synthesis Type II IFNs IFN-γ is secreted by TH1, CD8+ & NK cells IFN-γ function: Activates MQs Increases expression MHC Class I & II molecules; Increases antigen presentation Stimulate development of TH1 cells. IFN-γ is a potent immuno-regulatory cytokine. IFN-α and IFN-β are released from virus-infected cells soon after infection. These cytokines stimulate the NK cells, quickly leading to a rise in the NK-cell population from the basal level. The wave of NK cell activity peaks subsequent to this rise, about 3 days after infection. NK cells help contain the infection during the period required for the generation of CTLs. Once the CTL population reaches a peak, the virus titer rapidly decreases. Dr. Dekra El-Aghbary Associate Professor of Immunology 11 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 27 Inflammatory response Systemic inflammation Regulation of innate immunity Inflammatory response Inflammatory response or inflammation is the response of the immune system to an irritant such as bacteria, trauma, toxins, heat, which may lead to tissue injury. The requirements of acute inflammatory response Activation of vascular epithelium & increased vascular permeability Cytokines & other inflammatory mediators released in area of infection or damage Expression of selectin-type adhesion molecules on endothelial cells. Alterations in expression of cellular adhesion molecules (CAMs) & chemokine receptors facilitate movement of immune cells to sites of immune activity. Extravasation of inflammatory cells into infection area, Extravasation of inflammatory cells Neut. are first cells bind to inflamed endothelium & extravasation into tissues. Mono., MQ., & even eosin. may arrive 5–6 hours later in response to neut.- released mediators. Both of these events are signaled by recognition of PAMPs by PRRs. Extravasation of immune cells including phagocytes into area requires 4 sequential, overlapping steps: Step 1: Rolling Phagocytes attach loosely to endothelium by low-affinity, selectin CHO interactions. E-selectin molecules on endothelium bind to mucin-like adhesion molecules on phagocyte membrane 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 28 Step 2: Activation by chemoattractants Chemokines released in area during inflammation, such as IL-8, complement split product C5a, & N-formyl peptides produced by bacteria bind to Rs on phagocyte surface & trigger its activation. This signal induces a conformational change in integrin molecules in phagocyte membrane that increases their affinity for immunoglobulin- superfamily CAMs (Ig-CAMs) on endothelium. Step 3: Arrest & adhesion Interaction between integrins & Ig-CAMs mediates tight binding of phagocyte to endothelial cell & their movement through extracellular matrix. Step 4: Transendothelial migration Phagocyte extends pseudopodia through vessel wall & extravasates into the tissues. 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 29  Local mast cells within inflammatory environment, release other mediators including prostaglandins & histamines that add to vasodilation & capillary leakage & induce fever. Combination of vasodilation, capillary leakage, cytokine secretion, & movement of cells into damaged tissue gives rise to the four cardinal signs of inflammation-heat, redness, swelling, & pain. These local innate & inflammatory responses may be effective in clearing pathogens & skin wounds heal within hours or a few days. But when innate & inflammatory responses are not sufficient, perhaps because pathogens have evolved to evade innate responses, our powerful & last line of defense, adaptive immune responses come to rescue. DC (CCR7) has entered tissues & activated by binding to PAMPs via its PRRs, to nearest LN, where it can activate a T cell. Antigen-activated T cells then initiate adaptive immune responses against pathogen. Cytokines produced during innate immune responses support & direct adaptive immune responses to infection. Inflammation is a sign of an ongoing local innate immune response. However, sometimes localized tissue destruction that can accompany a vigorous immune 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 30  response itself becomes a problem & host then becomes a victim of chronic inflammation. Systemic inflammation A later component of inflammatory responses is acute phase response (APR), induced by proinflamatory cytokines (IL-1, TNF-, & IL-6). These cytokines have systemic effects on tissues, including fever, production of acute phase proteins (APP), & leukocytosis. APR is a term given to a collective of reactions to tissue injury resulting from infection, trauma, neoplasia, inflammation, and stress. The response is formulated by a number of different APP that vary in magnitude and type. The increased synthesis & secretion of APR by liver including mannose- binding lectin (MBL), C-reactive protein (CRP), & complement components, which act as antimicrobial proteins, activate a variety of processes that contribute to eliminating pathogens. Damage of vasculature together with vasodilation & increased vascular permeability, results in fluid loss into tissues that lowers blood pressure. These effects on blood vessels are particularly damaging to kidneys & lungs, which are highly vascularized. High circulating TNF-α & IL-1 levels also adversely affect heart. Thus, systemic inflammatory response triggered by septicemia can lead to circulatory respiratory failure, resulting in septic shock & death. 4 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 31 Regulation of innate immunity Many regulatory processes have evolved that either enhance or inhibit innate & inflammatory responses. The regulatory proteins control induction, type, & duration of these responses, in most cases resulting in elimination of an infection without damaging tissues or causing illness. Synthesis of APPs is enhanced by cytokines (TNF-α, IL-1β and IL-6) secreted by macrophages and endothelial cells. In addition to the major proinflammatory cytokines, C2a, C3a, C4a, & C5a, IFN production and the several APRs such as CRP, MBL and surfactant protein act as positive regulation for innate immunity. The change in plasma concentration of these proteins is accompanied by fever, leukocytosis, thrombocytosis, catabolism of muscle proteins and fat deposits. On the other hand, the anti-inflammatory cytokine IL-10 is produced late by M2 MQ. It inhibits production & effects of the inflammatory cytokines & promotes wound healing. Dr. Dekra El-Aghbary Associate Prof. of Immunology 5 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 32 Mechanism of killing by NK cells 1. Fas/FasL-mediated cytolysis The interaction of FasL with Fas on a target cell triggers apoptosis by Fas pathway. Fas pathway involved the association of Fas with the adapter molecule FADD, which in turn results in a series of reactions that activate a caspase cascade, leading to apoptosis of the target cell. The perforin-granzyme pathway. Granule exocytosis releases granzymes and perforin from the cytoplasm into the space between the NK cell and the target cell. Granzyme B enters the target cell by endocytosis and then passes into the cytoplasm through perforin pores. Granzyme B can cleave and activate the proapoptotic Bcl-2 family member Bid, which stimulates mitochondria to release cytochrome c. Cytochrome c, a molecule called Apaf, and procaspase- 9 assemble into an apoptosome, leading to caspase-9 activation and cleavage of procaspase-3, which activates death pathways. Granzyme B can also cleave and partially activate caspase-3. Release of cytochrome c and activation of caspase-3 are both required to initiate the caspase cascade that leads to target cell apoptosis. 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 33  Dr. Dekra El-Aghbary Associate Professor of Immunology 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 34  Complement System ❖ Complement components ❖ Major pathways of complement activation (initiation, components, cascade) ❖ Initiation of Complement by proteases ❖ Complement receptors ❖ Functions of Complement ❖ Regulation of Complement Activity ❖ Complement deficiencies Complement components Complement system comprises a group of serum proteins & glycoproteins. Most complement components are synthesized in the liver by hepatocytes, although some are also produced by blood monocytes, tissue macrophages, fibroblasts, and epithelial cells of the gastrointestinal and genitourinary tracts. Complement components (>50) constitute approximately 15% of the globulin protein fraction in plasma. In addition, since several of the regulatory components of the system exist on cell membranes, the term complement now embraces proteins and glycoproteins distributed between the blood plasma and cell membranes. Complement components are designated by numerals (C1–C9), by letter symbols (e.g., factor D, B). Also, complement proteins include Complement receptor (CR) proteins, & regulatory components. Some complement proteases become active by binding to other macromolecules & undergoing a conformational change; others are zymogens themselves, inactive until cleaved by another “upstream” protease. Smaller fragment resulting from cleavage of a component is designated “a” & larger fragment designated “b” (e.g., C3a, C3b; note that C2 is an exception: C2a is larger cleavage fragment). Larger fragments bind to target near site of activation, & smaller fragments diffuse from site & can initiate localized inflammatory responses by binding to specific receptors. They cooperate with both innate & adaptive immune systems to eliminate pathogens, dying cells, & immune complexes (ICs) from body. 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 35 The major pathways for complement activation are; classical, lectin, & alternative. C3 is a central component in all three complement activation pathways. The end result of initiating sequence of all three pathways is generation of C3 convertase & C5 convertase. Finally, all these pathways converge in a common sequence of events leading to membrane lysis. The sequence of proteins in a complement pathway from initiator protein to biological effector is referred to as a “complement cascade.” Classical pathway It is considered part of adaptive immune response since it begins with formation of antigen- antibody complexes. These complexes may be soluble, or they may be formed when an antibody binds to antigenic determinants (epitopes), situated on cell membrane of pathogen. Soluble antibody-antigen complexes are often referred to as immune complexes (ICs). Only complexes formed by antigens with IgM class or certain subclasses of IgG (IgG1 & IgG3) antibodies are capable of activating classical complement pathway. In serum, C1 exists as a macromolecular complex consisting of one molecule of C1q & two molecules each of serine proteases C1r & C1s, held together in a Ca-stabilized complex (C1qr2s2). Each C1 macromolecular complex must bind to at least two antibody constant regions for a stable C1q-antibody interaction to occur. An IgM molecule engaged in ICs can bind C1q, whereas circulating, non-antigen bound IgM cannot. C1q is can bind to CRP complexed with bacteria and can also bind tissue damage elements such as DNA, & histones on surface of apoptotic cells, resulting in opsonization & engulfment. 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 36 Initial activation involves interaction of ICs with complement components C1. C1q binds to an antibody, activating proteases, that cleaves C4 to C4a & C4b & C2 into C2a & C2b. C4b attaches to membrane & binds C2a forming C3 convertase (C4b2a) of classical pathway. C4b2a then combines with one molecule of C3b, forming C4b2aC3b called C5 convertase that cleaves C5 into C5a & C5b. Lectin pathway Lectin pathway is considered to be an arm of innate immunity. It is initiated When mannose-binding lectin (MBL, green) binds specifically to CHO on pathogens, activating MBL-associated serine proteases (MASPs, blue). MASPs cleave C4 & C2, generating C3 convertase (C4b2a). As for classical pathway, end result of initiating sequence of lectin pathway is generation of enzymes that cleave C3 into C3a & C3b, & C5 into C5a & C5b. MBL is constitutively expressed by liver. More recently, other lectin receptors have been recognized as initiators of lectin pathway. These include collectin-10 & collectin-11 as well as several members of ficolin family: ficolin-1, ficolin-2, & ficolin-3. Alternative pathway Initiation of alternative pathway, like lectin pathway, is independent of antibody-antigen interactions. Therefore, this pathway is also considered to be part of innate immune system. However, unlike lectin pathway, alternative pathway uses a different set of C3 & C5 convertases. up to 90% of deposited C3b molecules are generated via activation of 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 37 alternative pathway. All three initiation pathways culminate in formation of C5 convertase. For classical & lectin pathways, C5 convertase has composition C4b2a3b; for alternative pathway, C5 convertase has formulation C3bBbC3b. However, end result of all types of C5 convertase activity is same: cleavage of C5 molecule into two fragments, C5a & C5b. Large C5b fragment is generated on surface of target cell or immune complex & provides a binding site for subsequent components of membrane attack complex (MAC). Membrane attack complex (MAC) Activation of terminal components of complement cascade C5b, C6, C7, C8, & C9 results in deposition of a MAC onto microbial cell membrane. All of complement reactions take place on surfaces of microbial cells or on ICs in fluid phase of blood, lymph, or tissues, but MAC actually penetrates cell membrane. MACs introduce large pores in membrane, preventing it from maintaining osmotic integrity & resulting in death of cell. This complex can also form on infected host cells, although complement system must first overcome regulatory mechanisms designed to protect host cells from complement attack. 4 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 38 Initiation of Complement by proteases Alternative pathway can be initiated by some proteases such as thrombin & kallikrein that cleave C3 into C3b & Ca. Also. kallikrein can cleave B into Bb & Ba. Generation of C3a & C5a can also be effected by thrombin & plasmin cleavage of C3 & C5, linking coagulation & complement cascades. Interestingly, when blood platelets are activated during a clotting reaction, they release high concentrations of ATP and Ca2+ along with serine/threonine kinases. These enzymes act to phosphorylate extracellular proteins, including C3b. The Phosphorylated C3b is less susceptible to proteolytic degradation than its unphosphorylated form, & thus, by this route, activation of clotting cascade enhances all of complement pathways. Complement Receptors Complement receptor (CR) proteins on APCs and T cells surfaces bind complement proteins & signal specific cell functions. CR include CR1, CR2, CR3, CR4, CRIg….etc. 5 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 39 Functions of complement 1. Lysis of bacterial & cell membranes by MAC (C5b-C9). Neisseria meningitidis is a gram-negative bacterium that is susceptible to MAC-induced lysis, & patients who are deficient in any of complement components of MAC are particularly vulnerable to potentially fatal meningitis caused by these bacteria. 2. Enhancing phagocytosis Phagocytosis-enhancing components, C3b & C4b, serve as opsonins. The phagocytic cells are bind to these opsonins by many different CRs on their surfaces including CR1, CR3 & CR4 & CRIg; triggering phagocytosis of C3b-bound pathogen. 3. Induction of inflammation & chemotaxis by anaphylatoxin Some small complement fragments (C3a and C5a) act as inflammatory mediators. These 6 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 40 fragments bind to CRs on the endothelial cells lining small blood vessels and induce an increase in capillary diameter, thus enhancing blood flow to the affected area. Also, these fragments C3a and C5a, mediate degranulation of mast cells and basophils, resulting in release of mediators that induce contraction of smooth muscle, increased vascular permeability & attract other cells to the site of infection. Binding of anaphylatoxin complement component C5a to C5aRs on neut., stimulates degranulation & inflammation. They also attract other cells to site of tissue damage. Since these effects can be harmful (even lethal) in excess. 4. Clearance of circulating immune complexes Clearance of circulating ICs by binding to CRs on RBCs & removal of these complexes by CRs on MQs in liver & spleen. Deficiency in this process can lead to renal damage due to accumulation of ICs. 5. Augmentation of humoral immune response Stimulate production of antibodies by coligation of antigen to B cells via IgM & CD21. 7 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 41 B-cell coreceptor is a complex of three cell membrane molecules, CD21 (CR2), CD81 & CD19 which is important in antigen receptor signaling. C3d or C3dg, bind antigen to CR2, facilitating antigen-binding to B cell & stimulating an adaptive immune response to the same microbes. 6. Enhancement of antigen presentation by MBL, C1q, C3b, C4b, & C5a. 7. Clearance of apoptotic cells mainly by C1q. 8. Inducing IL production & aiding in cells activation. 9. Induction of Treg cells. Regulation of complement activation Several of regulatory components of system distributed between blood plasma & cell membranes. They limit effects of complement by promoting their degradation or preventing their binding to host cells. 1. C1INH (serine protease inhibitor) inhibits both C1rs of classical pathway & MASP of lectin pathway, inhibiting further activation of C4 & C2 & formation of C3 convertase. 2. Any C3 convertase complexes of either classical & lectin pathways or alternative pathway on host cells are degraded by host cell membrane protein decay-accelerating 8 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 42  factor (DAF). 3. Factor I & Membrane-cofactor protein (MCP) degrade C3b & C4b into inactive fragments only when it is associated on host cell membranes with necessary cofactors. 4. C5 convertase of alternative pathway, C3bBb3b, has a half-life of only 5 minutes, unless it is stabilized by reaction with Factor P (Properdin). 5. C5b component is extremely labile & is not covalently bound to membrane, as are C3b & C4b. Therefore, it is rapidly inactivated unless it is stabilized by binding of C6. 6. Protectin inhibits formation of MAC on host cells. 7. Lysis is usually minimized by binding to regulatory protein S & then destroyed. 9 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 43 Deficiencies of complement components Homozygous deficiencies in any of early components of classical pathway (C1q, C1r, C1s, C4, & C2) result in a marked increase in immune-complex diseases such as SLE, glomerulonephritis, & vasculitis. C1q has been shown to bind apoptotic cells & cell fragments. In absence of C1q binding, apoptotic cells can act as auto-antigens & lead to development of autoimmune diseases such as SLE. Individuals with deficiencies in early complement components may also suffer from recurrent infections with both gram-negative & gram-positive, pyogenic bacteria such as streptococci & staphylococci. These latter organisms are normally resistant to lytic effects of MAC, but early complement components are important in controlling such infections by mediating a localized inflammatory response & opsonizing bacterium. In particular, patients with deficiencies in C3 itself are unusually susceptible to infections with both gram-positive & gram-negative bacteria & immune-complex diseases. A deficiency in MBL results in serious pyrogenic infections in babies & children. MBL deficiency is also found with a frequency 2 to 3 times higher in patients with SLE than in normal subjects. Individuals with deficiencies in components of terminal complement cascade, factor D & properdin are suffer from Neisseria infections but not with immune-complex disease. Dr. Dekra El-Aghbary Associate Prof. of Immunology 10 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 44  MHC Molecules & Antigen Presentation Classes & Structure of MHC molecules The main differences between class I & II MHC molecules Role & Expression Patterns of the MHC Inheritance of MHC Major pathways of antigen processing & presentation Cross-presentation of exogenous & endogenous antigens Presentation of Non-peptide antigens Regulation of MHC expression Deficiency of MHC molecules Protein antigens must be processed into peptide fragments and presented by self- MHC molecules on the surface of host cells in order to be recognized by antigen- specific receptors (TCRs) on responding T cells. If antigen-presenting cells (APCs) are the link between innate & adaptive immunity, then MHC molecules are the tool that these cells use to create this link. MHC molecules hold antigenic fragments & present them to TCRs, allowing the associated T cell to become activated & initiating the first steps in the adaptive response. To associate with MHC molecules, antigens must first be cleaved into smaller fragments (processed) & transported to locations in the cell where they can bind with & stabilize MHC structure before they appear on cell surface (presentation). Classes of MHC Genes MHC genes are divided into three groups or classes: class I, II, & III. MHC class I region encode glycoproteins expressed on surface of nearly all nucleated cells; major function of class I gene products is presentation of endogenous (cytosolic) peptide antigens to CD8 T cells. In addition to the three genes that code for the polymorphic MHC class I molecules—HLA-A, HLA-B, & 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 45  HLA-C, also contains the much less polymorphic MHC class genes HLA-E, HLA-F, & HLA-G. They have a structure similar to the polymorphic class I gene products, but they do not present peptides to T cells. HLA-E & HLA-F are thought to be involved in presentation of antigens to NK cells. The expression of HLA-G by placental trophoblast cells has been suggested as a potential mechanism preventing rejection of fetus by maternal host. MHC class II genes encode glycoproteins expressed predominantly on APCs (MQs, DCs, & B cells), where they primarily present exogenous (extracellular) peptide antigens to CD4 T cells. They include pairs of genes that code for the polymorphic MHC class II molecules HLA-DP, HLA-DQ, & HLA-DR. Between the class I & class II regions is the class III region. MHC class III genes encode a diverse set of proteins, no structural or functional similarity to class I & II, some of which have immune functions, but that do not play a direct role in presenting antigen to T cells. This contains genes that code for serum complement components, C2, C4, & FB. The human MHC class III region also contains genes coding for the cytokines TNF-α & TNF-β (lymphotoxin-α). Structure of MHC molecules 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 46 Role of MHC Molecules To display a self-peptide in class I & II to test developing T cells for autoreactivity (in primary lymphoid organs) & maintain immune-tolerance to self-proteins (in secondary lymphoid organs). To display self-MHC class I & self-peptide to demonstrate that the cell is healthy. To display a foreign peptide in class I to show that the cell is infected & to engage with CTL cells. To display a foreign peptide in class II to show the body is infected & activate T cells. Expression patterns of MHC molecules MHC class I molecules are expressed on most nucleated cells, although expression levels can vary by cell type, and present pieces of proteins found in the cytosol of that cell; these proteins can be self or foreign in origin. While MHC class II molecules have a more limited distribution than MHC class I molecules; They are expressed constitutively only on APCs (B cells, MQs, & DCs) but can be induced on other cell types. DCs are considered the most powerful & most efficient of the pAPCs. These cells constitutively express high levels of MHC class II molecules & have inherent costimulatory activity, allowing them to quickly activate naïve T cells. 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 47  MQs must be activated before they express MHC class II molecules or costimulatory membrane molecules such as CD80/86. B cells express MHC class II molecules, although at low levels, & possess antigen- specific surface receptors. This makes them particularly efficient at capturing & presenting their cognate antigen, or the specific epitope recognized by their BCR. A variety of cells can function as APCs. Their distinguishing feature is their ability to express MHC class II molecules & to deliver a costimulatory, (second activating signal) to T cells. These non-profesional APCs include fibroblast (skin), thymic epithelia cells, glial cells (brain), thyroid epithelial cells, pancreatic beta cells, vascular endothelial cells. The levels of these proteins can fluctuate depending on local influences, such as viral infection (e.g., suppressed class I expression) & cytokine signaling (e.g., increased class II expression on pAPCs). Inheritance of HLA MHC class I & class II molecules differ from individual to individual within the population. These differences arise from: polygenicity, polymorphism, Codominant expression and Tightly linked 1. Polygenicity Polygenicity means that MHC class I & II molecules are coded by multiple independent genes. The human HLA complex contains three independent genes, HLA-A, HLA-B, and HLA-C, that code for MHC class I molecules. Similarly, the HLA complex codes for three different two-chain MHC class II molecules: HLA- DP, HLA-DQ, & HLA-DR. 2. Polymorphism Polymorphism means that multiple stable forms of each MHC gene exist in the population. The MHC is the most highly polymorphic gene system in the body & hence in the population: For example, in humans over a thousand slightly different alleles of the gene that codes for MHC class I molecule HLA-B have been identified. The extensive polymorphism of human MHC genes makes it very unlikely that two random individuals will express identical sets of HLA class I & class II molecules. Polymorphic residues in MHC alleles cluster in the peptide-binding 4 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 48  pocket, influencing the fragments of antigen that are presented to the immune system, and thereby influencing susceptibility to a number of diseases. 3, codominantly expression MHC genes show codominant expression patterns of both maternal and paternal copies. 4. Tightly linked Due to the tightly linked, set of alleles within entire MHC locus is generally passed down as one unit; one such linked set of MHC alleles inherited from a parent is called a haplotype. Moreover, a new haplotype, R (recombinant), can arise from rare recombination of a parental haplotype (maternal shown here). The HLA haplotypes of a son or daughter differs from the haplotypes of his or her parents: Each parent expresses one HLA haplotype different from each child. Every child in the family expresses a different set of HLA haplotypes, but there is a 1 in 5 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 49  4 chance that any two siblings will have identical HLA haplotypes. However, individuals within a family do not usually express identical MHC molecules, & the likelihood of finding individuals in the general population who are matched at all HLA alleles is extremely small. The MHC haplotype will influence both the specific peptides that can be presented as well as the T-cell repertoire in an individual, shaping and sometimes limiting the ways in which foreign antigen can be recognized by that host. Species diversity at the MHC locus imparts an evolutionary survival advantage against mortality from infectious disease. Major pathways of antigen processing & presentation There is at least two distinct routes of antigen processing & presentation that differ in their source of antigen, intracellular trafficking, & MHC association. In general, antigens from intracellular sources are presented in MHC class I molecules (endogenous pathway) to CD8 T cells while antigens from extracellular spaces are presented in MHC class II molecules (exogenous pathway) to CD4 T cells. Endogenous (Cytosolic) Pathway While all cells have constitutive proteasomes involved in the processing and presentation of cytosolic proteins in MHC class I molecules, infected cells or pAPCs can temporarily express immunoproteasomes, which generate peptide fragments that are optimized for MHC class I binding. This immunoproteasome induced by exposure to certain cytokines, such as IFN-γ or TNF. 6 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 50  The intracellular proteins are degraded into short peptides by cytosolic proteasomes/immunoproteasomes. Peptides are selectively transported from the cytoplasm into the rough endoplasmic reticulum (RER). In RER, peptides bind to newly synthesized MHC class I molecules, (α & β2m chains) which are synthesized separately on ribosomes. A peptide that binds to an MHC class I molecule in RER moves via the Golgi apparatus to the cell surface, where it is displayed & presented to a CD8+ T cell expressing the appropriate antigen receptor. Exogenous (endocytic) pathway Exogenous antigens are antigens that come from outside a host cell & are taken inside, normally by endocytosis or phagocytosis. They can be derived from pathogens (bacteria or viruses) or from foreign proteins (vaccines) that do not injure the host but activate an immune response. The protein is internalized, contained in an intracellular vesicle that fuses with endosomal or lysosomal vesicles that are highly acidic (pH ~4.0). These vesicles contain an array of degradative enzymes, including proteases as cathepsins & peptidases. The MHC class II α & β chains are synthesized individually in RER which leave the ER & enter the Golgi complex, & from there they proceed into the acid vesicle endocytic pathway. Vesicles containing MHC class II fuse with acid vesicles (endosomes or lysosomes) containing peptides derived from the catabolism of exogenous antigens. Ultimately class II molecules reach the cell surface & are presented on the membrane to CD4 TH cells. In most cases, class I molecules present processed endogenous (intracellular) antigen to CD8 T cells and class II molecules present processed exogenous (extracellular) antigen to CD4 T cells. However, in some cases, exogenous antigens internalized by DCs can gain access to the endogenous presentation pathway in a process called cross-presentation, leading to peptide association with MHC class I and engagement with CD8 T cells. Cross-Presentation In addition to their ability to process exogenous antigens in the MHC class II pathway, APCs & particularly DCs, have a unique pathway, called cross- presentation, for generating peptides derived from exogenous protein antigens & presenting them to CD8+ T cells. The DC takes up exogenous antigens (such as those derived from a virus-infected or dying cell) by either phagocytosis or 7 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 51  pinocytosis. These peptides are transported into the ER where they bind to newly synthesized MHC class I molecules & thus activate naïve CD8+ T Cells. Ligand binding to PRRs, especially TLRs, can induce maturation of DCs as well as trigger the transport MHC class I molecules from intracellular stores to these antigen- laden vesicles arising from extracellular sources. The ability of DCs to cross-present antigens from extracellular sources has great advantage for the host. It allows these APCs to capture antigen, such as viral proteins, from extracellular environment or from dying cells, process these antigens, & activate CTLs that can then seek out & attack virus-infected cells, inhibiting further spread of the infection. In some instances pAPCs will divert endogenous antigen of infected pAPCs to a pathway that leads to MHC class II loading & peptide presentation to CD4 T cells. Likewise, autophagy, the homeostatic process whereby cytosolic components become enclosed in vesicles that traffic through lysosomal compartments, can result in cytosolic peptides being presented by MHC class II molecules. Self- peptides can bind to MHC class II in these acid vesicles. Presentation of non-peptide Antigens Lipids & glycolipids are presented by a small group of structurally similar proteins encoded outside MHC locus: a group of non-classical class I molecules, including CD1 family of proteins & MHC class I–related protein (MR1). Five human CD1 genes & one MR1 gene have been identified. They share structural similarity with class I, however, CD1 & MR1 display very limited polymorphism. Functionally, CD1 & MR1 molecules resemble MHC class II proteins, moving intracellularly to endosomal compartments, where they associate with exogenous antigen. They present non-protein antigens to αβ & γδ T cells and serve to regulate immune homoeostasis as well as control some infectious agents at mucosal surfaces. Regulation of MHC expression At the levels of genes, MHC genes are regulated separately, because MHC class I molecules can be expressed in the absence of MHC class II molecules. A class I MHC transcriptional activator called CITA (Class I transactivator) has been shown to activate the promoter of class I MHC genes while CIITA (Class II 8 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 52  transactivator) & another transcription factor called RFX, have both been shown to activate the promoter of class II MHC genes. However, at the levels of external regulation, expression of MHC molecules is coordinately regulated, meaning that all classes I & II molecules are expressed at the same time on a single cell. Cytokines released during the response to infectious agents enhance expression of MHC molecules, thus enhance T-cell responses: Factors such as IFN-γ enhance expression of all class I & II molecules on a particular cell while IFN α, and β upregulate MHC class I expression. IFN-γ has been shown to induce expression of the CIITA, thereby indirectly increasing expression of class II MHC molecules on a variety of cells, including non-APCs (e.g., Fibroblast, skin keratinocytes, intestinal epithelial cells, vascular endothelium, placental cells, & pancreatic beta cells). However, IL-4 increases expression of class II molecules in resting B cells, turning them into more efficient APCs. Conversely, expression of class II molecules by B cells is down-regulated by IFN-. Viral interference, one clear example of negative regulation of MHC comes from viruses that interfere with MHC class I expression & thus avoid easy detection by CD8 T cells. These viruses include human CMV, HBV, & Ad12. For example, in CMV infection, a viral protein binds to ß2m, preventing assembly of class I MHC molecules & their transport to plasma membrane. Also, HSV-1 downregulates expression of CD1d, inhibiting glycolipid antigen presentation. In addition, tumor cells frequently show decreased expression of MHC class I molecules compared to normal cells, so reducing a potential antitumor response by CD8+ T cells. Corticosteroids & prostaglandins can also decrease expression of class II MHC molecules. Deficiencies in MHC expression Rare people with bare lymphocyte syndrome (BLS) lack the ability to express either HLA class I or II molecules or both. In early life, this individual suffers frequent bacterial infections of upper RT & in the second decade begins to experience chronic infection of lungs. 9 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 53 MHC Alleles & Susceptibility to Certain Diseases Some HLA alleles occur at a much higher frequency in people suffering from certain diseases than in the general population. The diseases associated with particular MHC alleles include autoimmune disorders, certain viral diseases, disorders of the complement system, some neurologic disorders, and several different allergies. One important recent example is the association described between HLA-C & progression to AIDS in HIV infection: higher expression of HLA-C is associated with reduced viral load & reduced rate of progression to low CD4+ T cell counts. The association between high HLA-C expression & improved control of HIV infection may result from a more effective CD8+ T-cell response to the virus in these individuals than in others who do not express high levels of HLA-C. Dr. Dekra El-Aghbary Associate Professor of Immunology 10 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 54  Ontogeny of Thymocyte Development of Thymocyte in the Thymus Thymic selection of thymocytes Mechanisms of self-tolerance T-cell activation and differentiation Signals for T-cell activation Activation by superantigens Clonal anergy versus clonal expansion Development of Thymocyte in the Thymus HSC from the bone marrow travel to the thymus via the bloodstream, undergo development to mature T cells, and are exported to the periphery, where they can undergo antigen-induced activation and differentiation into effector cells and memory cells. Each stage of development occurs in a specific microenvironment of the thymus and is characterized by specific intracellular events and distinctive cell-surface markers. Immature T cells are called thymocytes, and progress through several stages of development defined broadly by the expression of the coreceptors CD4 and CD8. T-cell development is divided into two phases: a. Early thymocyte development which is largely T-cell receptor-independent. The specific events in this early phase (double negative, DN) include the following: 1. Commitment of hematopoietic precursors to the T-cell lineage, 2. T-cell receptor (TCR) gene rearrangements, and 3. Expansion of cells that have successfully rearranged TCR genes. b. The second phase of T-cell development is largely dependent on TCR interactions (double positive, DP). The events include the following: 1. Thymic selections (positive and negative selection), and 2. Lineage commitment in which the thymocytes give rise to effector cell lineages, including CD4+ helper or CD8+ cytotoxic populations, as well as CD4+ regulatory T cells. Cellular organization of the thymus T-cell precursors enter the thymus via blood vessels at the corticomedullary junction and then travel into the cortex, proliferating first in the region just below the capsule (the subcapsular cortex). As they mature, they migrate from the cortex into the medulla and ultimately exit via vessels in the corticomedullary junction. The most immature, CD4-CD8- (DN) thymocytes pass through several stages (DN1-DN4) defined by CD44, 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 55 CD25, & c-Kit expression, during which they commit to the T-cell lineage and begin to rearrange their TCR gene loci. Those that successfully rearrange their TCR β chain proliferate, initiate rearrangement of their TCR α chains, and become CD4+ CD8+ (DP) thymocytes, which are the most abundant subpopulation in the thymus & the first cells to express a fully mature TCR αβ/CD3. DP thymocytes undergo positive and negative selection in the thymic cortex. Positively selected DP thymocytes must decide whether to become helper CD4+ T cells or cytotoxic CD8+ T cells. This process, called lineage commitment, depends on the kinetics of TCR signaling experienced by DP thymocytes. Positively selected thymocytes continue to mature and up-regulate the CCR7 chemokine receptor, which allows them to migrate from the thymic cortex to the thymic medulla. In the medulla, they are subject to another round of negative selection to self- antigens that include tissue-specific proteins. Single positive (SP), CD4+ or CD8+ cells initiate a cellular program that enhances their survival and up-regulates receptors (IL-7R & CCR7) that facilitate their migration from the thymus and through the blood to the lymph nodes. The exit of SP thymocytes from the thymus depends on expression of the sphingosine 1-phosphate receptor 1 (S1P receptor 1) and its interaction with S1P at the corticomedullary junction. Mature thymocytes that have just left the thymus are referred to as recent thymic emigrants. They are not yet optimally functional and undergo a post-thymic phase of maturation in secondary lymphoid tissue that fully licenses them as naïve T cells. 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 56 Most thymocytes become TCR-αβ T cells. However, TCR-γδ cells are the first T cells to mature during fetal development and populate the periphery in several early waves. TCR-γδ and TCR-αβ cells are functionally distinct. Most TCR-γδ cells have limited receptor diversity and antigen specificity. They are very important first responders to pathogens at mucosal surfaces and the skin. TCR-αβ T cells have more diverse specificities, anatomical ranges, and functions. In other hand, a small percentage of DP thymocytes, high-affinity TCR interactions do not lead to deletion, but instead drive development to specialized cell lineages, including natural killer T (NKT) cells, intraepithelial lymphocytes (IELs), and regulatory T cells (Treg) each of which has a distinct function. Thymic selection of thymocytes Thymic selection involves multiple interactions of DP and SP thymocytes with both cortical and medullary thymic stromal cells, as well as DCs and MQs. Selection results in a mature T-cell population that is both self-MHC restricted and self-tolerant. Thymic selection includes two distinct selection processes: Positive selection, which selects for those thymocytes bearing receptors capable of binding self-MHC molecules with low affinity, resulting in self-MHC restriction; the property of recognizing antigenic peptides only in the context of self MHC molecules. Positive selection occurs in the cortex. Cortical thymic epithelial cells (cTECs) express a distinct proteasome system that produces and presents a unique set of peptides for positive selection. The signaling protein, Themis which is expressed specifically by thymocytes and appear to be required for positive selection. In general, DP thymocytes have one of three fates depending on the affinity of their TCR for self-MHC/self-peptide complexes expressed by thymic stromal cells. The large majority of DP thymocytes die in the cortex by neglect because of their failure to bind MHC/peptide combinations with sufficient affinity. The small percentage (2% to 5%) whose TCRs bind MHC/peptide with high affinity, particularly on the surface of DCs, die by clonal deletion (negative selection). Those DP thymocytes (2%-5%) whose receptors bind to MHC/peptide with low to intermediate affinity are positively selected and mature to single-positive (CD4+ or CD8+) T lymphocytes (lineage commitment). Transcription factors Th-POK and Runx3 play key roles in CD4 helper cell and CD8 cytotoxic cell commitment, respectively. Positively selected thymocytes up-regulate the CCR7 chemokine receptor, which allows them to migrate from the thymic cortex to the thymic medulla. Negative selection, which selects against thymocytes bearing receptors with high affinity for self-MHC/self-peptide complexes, resulting in self-tolerance, unresponsiveness to self-antigens. Negative selection can occur in both the cortex and medulla of thymus and is mediated by thymic APCs, DCs and B cells. Furthermore, SP migrate to medulla and can be negatively selected for tissue-specific antigens presented by medullary epithelial cells (mTECs) which express high levels of costimulatory ligands (CD80 & CD86). mTECs express also the transcription factor AIRE (autoimmune regulator), which is in large part responsible for their unique capacity to express tissue specific- antigens. Medullary DCs can acquire antigens from mTECs and also mediate negative selection. A mutation in AIRE gene caused autoimmune polyendocrinopathy syndrome 1(APS1). 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 57 Mechanisms of self-tolerance The mechanisms that maintain self-tolerance can be central and peripheral tolerance. Central tolerance They include clonal deletion (thymic negative selection), clonal arrest where thymocytes that express autoreactive T-cell receptors are prevented from maturing; clonal anergy, where autoreactive cells are inactivated, rather than deleted; and clonal editing, where autoreactive cells are given a second or third chance to rearrange a TCR α gene; and generation of regulatory T cells. A fraction of thymocytes that experience high-affinity TCR interactions do not die by negative selection but develop into FoxP3 Treg cells that inhibit T-cell responses outside the thymus. These cells require help from cytokines, including IL-2 and IL-15, to complete their maturation. Treg cells that 4 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 58 develop in the thymus are called thymic Treg cells while Treg cells that differentiate in the periphery are called peripheral Treg cells. The function and location of both Treg populations overlap, although they may have distinct responsibilities in different tissues. Peripheral Tolerance Not all self-reactive lymphocytes are deleted by central tolerance due to the absence of some self-antigen in thymus and because some weakly self-reactive T-cell are surviving and escape from thymus. The mechanisms of peripheral tolerance enforce T-cell tolerance in the periphery. Peripheral tolerance induced when mature T cells recognize self-antigen in peripheral tissue. This involves: 1. Death by Activation-induced cell death (AICD) via coexpression of death receptor (DR), such as Fas/FasL & TNF/TNFR and production of proapototic proteins such as Bad and Bax. 2. Anergy (functional inactivation) 3. Suppression by T reg cells Treg inhibit immune responses in several ways; (1) Cytokine deprivation: T s express relatively high levels of high-affinity IL-2 receptors and can compete for the cytokines that activated T cells need to survive and proliferate. (2) Cytokine inhibition: They secrete several cytokines, including IL-10 and TGF-β, which bind receptors on activated T cells and reduce signaling activity. (3) Inhibition of APCs: Treg can interact directly with MHC class II–expressing APCs and inhibit their maturation, leaving them less able to activate T cells. (4) Cytotoxicity: Treg can also display cytotoxic function and kill activated cells directly by secreting perforin and granzyme. T-cell activation and differentiation Activation of a naïve T cell in the secondary lymphoid tissues results in the generation of effector and memory T cells. This activation requires several receptor-ligand interactions between the T cell and a DC, as well as signals through cytokines produced by the activating APC and other supportive cells in the lymphoid organ. CD4+ T cells become effector helper T cells (TH) and secrete cytokines that enhance the activity of many other immune cells. CD8+ T cells become cytotoxic T cells (Tc) that kill infected cells. 5 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 59 Signals for T-cell activation Three distinct signals are required to induce naïve T-cell activation, proliferation, and differentiation. Signal 1 is triggered by TCR-CD3 complex/MHC-peptide interaction, along with CD4 and CD8 coreceptors and adhesion molecules. Adhesion molecule interactions, two of which (LFA-1/ICAM-1, CD2/LFA-3) are, markedly strengthen the connection between the T cell and APC or target cell so that signals can be sustained; Signal 2 is generated by interactions between specific costimulatory receptors on T cells and costimulatory ligands that are expressed only by pAPCs, such as CD28; and Signal 3 provided by local cytokines, that directs the differentiation of T cells into distinct effector cell types, which required for the full T-cell activation. Several different pAPCs can provide Signals 1, 2, and 3 to a naïve T cell, although DC subsets appear particularly potent activator of naïve T cell. 6 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 60 Activation by superantigens Superantigens are a special class of T-cell activators. They are products of certain strains of Staphylococcus aureus (Staphylococcus aureus toxic shock toxin (TSST-1), group A Streptococcus, or other microbes that are released extracellularly as toxins and act as nonspecific polyclonal T cell mitogens and cytokine inducers. Superantigens are not processed by APCs but, rather, are presented as intact proteins by MQs to T cells. They simultaneously bind to MHC class II and the β chain of the TCR on CD4+ T cells. This crosslinking of the TCR and MHC class II mimics signal 1 and activates T cells; signals 2 and 3 are provided by the MQ. This interaction activates all T cells expressing these chains, regardless of the TCR α chain they express and their antigen specificity. A particular superantigen can activate 5–20% of an individual’s total T cells, in contrast to the ability of a peptide+MHC complex to activate less than 0.01% of T cells. Superantigens induce massive production of cytokines, particularly TNF, IL-1, and IL-6. This cytokine storm can result in serious consequences with systemic toxicity, including fever, skin peeling, shock and even death. Human populations vary in their susceptibility to superantigens due to polymorphisms in MHC class II, which influences the binding of the superantigen. Superantigens are exogenous and endogenous. Exogenous superantigens are soluble secreted bacterial proteins, including various exotoxins. Endogenous superantigens are membrane-embedded proteins produced by certain viruses. Clonal anergy versus clonal expansion\ a) Resting T cells that engage antigen and costimulatory ligands CD80/86 are activated and expand. b) T cells that engage antigen on the surface of a non-APC (such as a pancreatic islet beta cell) will not receive costimulatory signals and, instead, are inactivated or anergized. c) Anergy can also be induced when a T cell engages antigen and receives inhibitory rather than costimulatory signals through coinhibitory receptors such as CTLA-4 and PD-1. PD-1 and CTLA-4 are coinhibitory receptors that block T-cell activation, acting as checkpoint that down-regulate immune responses; up-regulated by T cells after activation. 7 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 61 Signals that lead to clonal anergy versus clonal expansion Dr. Dekra El-Aghbary Associate Professor of Immunology 8 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 62  Effector response of T cells Cell-Mediated immunity TH cell subset polarization Effector response of TH cells Cross-regulation of T helper cell subsets Cytotoxic T cell subsets Effector response of Tc cells Generation of memory cell subsets Cell-Mediated Effector Responses T cell-mediated immune responses can be divided into two major categories according to the different effector populations that are mobilized: cytotoxic T cells and helper T cells. TH cells exert their effector functions, by contributing to the activation and regulation of APCs, B cells, and cytotoxic T cells via receptor-ligand interactions and soluble cytokines and chemokines. On the other hand, cytotoxic cells exert their effector functions directly, by attacking infected cells, abnormal cells and, in some cases, the pathogens themselves. Effector cytotoxic cells arise from both the adaptive and innate immunity. Innate immune cells that contribute to the clearance of infected cells include NK cells and myeloid cell types such as MQs, neutrophils, and eosinophils. Adaptive cytotoxic cells include CD8+ cytotoxic T lymphocytes (CTLs or Tc cells), as well as the CD4+ NKT cell subpopulation, which, although derived from the T-cell lineage, displays some useful features of innate immune cell types, too. Even some populations of CD4+ T cells can be cytotoxic, producing cytokines that cause cell death. CTL, NKT, and NK effectors all induce cell death by triggering apoptosis in their target cells. Not only do these cytotoxic effector cells eliminate targets infected with intracellular pathogens (viruses and bacteria), but they also play a critical role in eliminating tumor cells and cells that have been stressed by extreme temperatures or trauma. CTL and NK cells also play a less desirable role in rejecting cells from allogeneic organ transplants. Essentially, the cell-mediated immune response is prepared to recognize and attack any cell that exhibits “nonself” or “altered-self” characteristics. 1 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 63 The humoral and cell-mediated immune systems also cooperate effectively. Cells such as MQs, neutrophils, eosinophils, and NK cells all express Fc receptors, which can induce phagocytosis of antibody-antigen complexes and/or the direct killing of target cells via a process known as ADCC. TH cell subset polarization Signal 3 also includes another important set of cytokines, known as polarizing cytokines. These are produced by a variety of cell types, including APCs, T cells, and innate lymphoid cells (ILCs), and play central roles in determining what types of effector cells naïve T cells will become. The interaction of pathogen with PRRs on DCs and other neighboring immune cells determines which polarizing cytokines are produced and, hence, into which T helper subset a naïve T cell will differentiate. In general, polarizing cytokines that arise from DCs or other neighboring cells interact 2 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 64 with cytokine receptors and generate signals that induce transcription of unique master gene regulators. These master regulators, in turn, regulate expression of various genes, including effector cytokines, which define the function of each subset. CD4+ T cells differentiate into at seven subpopulations of effector cells: TH1, TH2, TH9, TH17, TH22, peripheral regulatory T cell (pTreg), and (follicular T helper cell) TFH cells. Each subpopulation is characterized by (1) a unique set of polarizing cytokines that initiate differentiation, (2) a unique master transcriptional regulator that regulates the production of helper-cell-specific genes, and (3) a distinct set of effector cytokines that they secrete to regulate the immune response. The different helper T- cell subsets deliver effector cytokines that are tailored to the pathogen that initiated the immune response. Cytokine milieu (signal 3) determines CD4 T cell subset polarization. Signals 1 and 2 from the APC cell activate the CD4+ T cell. Signal 3 polarizes the T cell to an effector cell subset that is defined by its cytokine profile. CD4+ Th1 cells regulate the response to intracellular pathogens, including viruses. TH2 and TH9 cells regulate our response to worms (helminths). TH17 cells regulate responses to fungi and extracellular bacteria, particularly at our barrier organs. TH22 cells may contribute to the inflammation associated to with autoimmune disorders. 3 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 65 TFH cells are responsible for helping B cells during affinity maturation in the germinal centers. PTreg cells inhibit T-cell responses and help quell autoimmunity. Effector responses of TH cells Although each T-cell effector subset is unique, they share some functions with other cells, including ILCs, and work together to mount responses that are divided into two major categories. Type 1 responses to viruses and many bacteria involve the activation of effector subsets that coordinate cytotoxic responses. Type 2 responses to worms, protozoa, and allergens involve the activation of effector subsets that coordinate IgE and eosinophilic responses. Helper T cells can provide direct, cognate help to B cells and influence their differentiation via expression of CD40L and the secretion of effector cytokines. Also, they can provide indirect help to CTLs and other neighboring immune cells by interacting with APCs and producing cytokines that influence cytotoxic and inflammatory activity of multiple cell types. Helper T-cell subsets can also exacerbate inflammatory diseases and can participate in autoimmunity and allergy. Cross-regulation of T helper cell subsets Helper T-cell subsets often “cross-regulate” each other. The cytokines they secrete typically enhance their own differentiation and expansion, while inhibiting commitment to other helper T-cell lineages. Cross-regulation of T helper cell subsets by transcriptional regulators; GATA-3 and T-Bet reciprocally regulate differentiation of TH1 and TH2 lineages. IL-12 promotes the expression of the TH1-defining transcription factor, T-Bet, which induces expression of TH1 effector cytokines, including IFN-γ. At the same time, T-Bet represses expression of the TH2-defining master transcriptional regulator, GATA-3, as well as expression of the effector cytokines IL-4 and IL-5. Reciprocally, IL-4 promotes expression of GATA-3, which up-regulates the synthesis of IL-4 and IL-5, and at the same time represses expression of T-Bet and the T 1 effector cytokine IFN-γ. 4 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 66 Cytotoxic T cell subsets Effector CD8 cytotoxic cells are not as diverse as CD4 effector T helper cells, but can develop into two distinct subsets: Tc1 cells and Tc2 cells. These subtypes a loosely resemble TH1 and TH2 cells in terms of the cytokines they generate as well as the cytokines that promote their development. CTLs are biased toward becoming Tc1 cells, which secrete IFN-γ but not IL-4. In the presence of IL-4, CTLs develop into Tc 2 cells, which secrete much more IL-4 and IL-5 than IFN-γ. Both subsets are potent killers, although Tc1 cells can use both perforin/granzyme- and FasL-mediated strategies, whereas Tc2 cells appear to use only perforin and granzymes. Effector Responses of cytotoxic T cells A CTL can kill a target in two major ways: either via the directional release of granule contents, or by a Fas-FasL membrane signaling interaction. Rather than inducing cell lysis, both of these processes induce the target cell to undergo apoptosis, typically within an hour of contact with the cytotoxic cell. 1. Granzyme- and perforin-mediated cytolysis Many CTLs initiate killing of their targets via the delivery of proapoptotic molecules. These molecules are packaged within granules that can be visualized by microscopy. Analysis of their contents revealed 65-kDa monomers of a pore-forming protein called perforin and several serine proteases called granzymes. Formation of a CTL- 5 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 67 target cell conjugate is followed within several minutes by a Ca-dependent, energy- requiring step in which, ultimately, the CTL induces death of the target cell. The CTL then dissociates from the target cell and may go on to bind another target cell. Although granzyme B was once thought to gain entry into the target cell via surface perforin pores, it is now thought that it enters mostly via endocytic processes. Many target cells express the mannose 6-phosphate receptor on their surface, which binds granzyme B. Complexes of granzyme B bound to mannose 6-phosphate receptor are internalized and appear inside endosomal vesicles. Granzyme proteases do not directly mediate DNA fragmentation. Rather, they activate an apoptotic pathway within the target cell. Within several minutes of CTL contact, target cells begin to exhibit DNA fragmentation. CTL-mediated killing not only kills virus-infected cells but also can destroy the viral DNA in those cells directly. The rapid onset of DNA fragmentation after CTL contact may prevent continued viral replication and assembly in the period before the target cell is destroyed. 2. Fas/FasL-mediated cytolysis Some potent CTL lines have been shown to lack perforin and granzymes. In these cases, cytotoxicity is mediated by Fas (CD95). This transmembrane protein, expressed by many cell types, is a member of the TNF receptor family and can deliver a death signal when cross-linked by its natural ligand, a member of the TNF family called Fas ligand (FasL). The interaction of FasL with Fas on a target cell triggers 6 ‫ﻣﻊ ﲢﻴﺎﺕ ﻣﺮﻛﺰ ﺍﳋﻠﻴﺞ ﺍﻟﻌﺮﺑﻲ‬ 68 apoptosis. Fas mutations lead to multiple disorders in humans as autoimmune lymphoproliferative syndrome (ALPS or Canale-Smith syndrome). NKT Cells A third type of cytolytic lymphocyte has been identified with characteristics shared by both the CTL and the NK cell. This cell type, designated the NKT cell to reflect its hybrid quality, develops in the thymus, and it is a member of the adaptive immune system. It undergoes antigen-receptor gene rearrangements and expresses an αβ TCR complex on its surface. However, it also exhibits characteristics of cells in the innate immune system. The TCR on many human NKT cells is invariant (very limited diversity), therefore sometimes referred to as invariant NKT (iNKT) cells. The TCR on NKT cells does not recognize MHC-bound peptides but rather glycolipid antigens presented by the non-polymorphic MHC class I—related CD1 molecule. Finally, NKT cells also appear to contribute to viral immunity, despite the fact that viruses do not typically express glycolipids. NKT cells exhibit both helper and cytotoxic activity and kill cells predominantly via FasL-Fas interactions. Moreover, they may play an indirect role in shaping the viral immune responses via the production of cytokines, including IFN-γ, IL-2, TNF-α, and IL-4. Cytotoxic cells Cell type Effector molecules produced Mechanism of killing CTL Cytotoxins (perforin & granzyme), IFN-γ, TNF, Cytotoxic granules release, (typically FasL Fas/FasL interactions CD8+) NKT cell IFN-, IL-4, GM-CSF, Il-2, TNF, FasL Fas/FasL interactions