BBS3014 Immune Responses in Health and Disease Course Summary PDF

 BBS3014 – Małgorzata Maciążek BBS3014 Immune responses in health and disease Małgorzata Maciążek 1  BBS3014 – Małgorzata Maciążek...

 BBS3014 – Małgorzata Maciążek BBS3014 Immune responses in health and disease Małgorzata Maciążek 1  BBS3014 – Małgorzata Maciążek Contents...........................................................................................................................................1 Introduction to immunology...............................................................................................3 Innate immune system.......................................................................................................5 Substances released by innate immune cells................................................................6 Physiological barriers of innate immune system............................................................7 Development process of the immune cells:...................................................................8 Release of the immune cells from the bone marrow......................................................8 Migration of the immune cells........................................................................................9 Pattern recognition receptors....................................................................................... 11 Neutrophils................................................................................................................... 15 Macrophages................................................................................................................ 18 Complement system.................................................................................................... 22 Innate lymphoid cells................................................................................................... 26 NK cells......................................................................................................................... 30 Dendritic cells.............................................................................................................. 37 MHC molecules............................................................................................................ 42 Adaptive immune system................................................................................................. 47 T cell development – T lymphopoiesis.......................................................................... 49 T cells – all information................................................................................................. 59 B cell development....................................................................................................... 82 B cells – general information........................................................................................ 87 Ageing of the immune system........................................................................................ 105 Workshop lectures......................................................................................................... 108 Vaccination................................................................................................................. 108 Transplantation.......................................................................................................... 115 Obesity....................................................................................................................... 124 Autoimmune diseases................................................................................................ 129 2  BBS3014 – Małgorzata Maciążek Introduction to immunology Immunology - scientific discipline that studies how the body defences itself against injuries, infectious organisms or foreign compounds 2 types of immune system: innate and adaptive characteristics innate adaptive specificity for molecules shared by for many different microbial groups of related microbes and non-microbial antigens and molecules produced by damaged host cells diversity low - molecules encoded by very high - antigen receptors inherited genes are generated by somatic recombination of gene segments in lymphocytes memory limited yes cellular and chemical skin, mucosal epithelia, lymphocytes in epithelia, barriers antimicrobial molecules antibodies secreted at epithelial surface secreted proteins complement, various lectins antibodies cells phagocytes (macrophages, lymphocytes: B cells and T neutrophils) dendritic cells, cells NK cells, mast cells, innate lymphoid cells 3 phases of immune response: 1. infection → pro-inflammatory pathways, inflammation and clearance pathogen replicates fast, which causes fast response from innate immune system: → rapid response to broad classes of pathogens → controls pathogen replication while stimulating adaptive immunity 3  BBS3014 – Małgorzata Maciążek innate immune system → not enough for full pathogen clearance → adaptive immunity: → slow initial response to a specific pathogen → its robust primary response often required for pathogen clearance innate immunity - dies down as quickly as it got activated, however adaptive immunity does not go to 0 due to memory cells 2. resolution → anti-inflammatory pathways, return to homeostasis adaptive immune system → maintains at a certain level = pool of memory cells after the first infection 3. immune memory → memory cells are produced and kept safe, so the body now how to react wicker and better during re-infection durable protection → if reinfection: innate immunity happens the same way as the first infection, however the adaptive immune system responds starts as quickly as innate immune response but even more powerful than before → due to memory cells what do you need for successful immune response: 1. recognize pathogens → receptors 2. changes in response to infection (activation of immune cell) 3. make effector mechanisms that kill pathogens → cytokines, chemokines and all signalling molecules = contribute to killing in 2 ways: → directly (killing, digesting enzymes) – direct cytotoxicity → non-directly (signalling, communicative molecules) – indirect communication with other immune cells 4. resolve inflammation and return to homeostasis → self-downregulation 5. *Only for some: form memory cells that protect against future infections – determined in step 3 by specific gene expression 4  BBS3014 – Małgorzata Maciążek Innate immune system Natural/naïve - present at birth, inherited, first line of defence, antigen-nonspecific defence mechanism used immediately or within hours after exposure to a microbe How it works: by inflammation – recruiting phagocytes and other leukocytes that destroy microbes starting on the clean-up pathogens start of repairing the barriers again/clean-up of damaged issues barriers Components: physical (epithelial cells), chemical (compounds in swat, tears, saliva – low pH) and physiological (normal commensal bacterial microflora) barriers phagocytic cells (neutrophils, macrophages), dendritic cells (DCs), mast cells, natural killer (NK cells), and other innate lymphoid cells; blood proteins, including components of the complement system and other mediators of inflammation creating signals for adaptive immune system Cell type: General information: Function: Neutrophils Granulocyte “swallow” microorganisms, 2 types: band neutrophil and encased in phagosome -> segmented neutrophil granules release enzymes Amoeboid motion + from the inside chemotaxis First cells to arrive at the scene of infections Eosinophil Granulocyte Defence against parasites – Motile, phagocytic toxic proteins release Enzymatically degrade walls Activated by cytokines or of pathogens allergens coated with IgE Basophils Agranulocyte, leukocyte Influences adaptive immune Granules with histamine and responses + exert tissue hydrolytic enzymes repair function Monocytes: Agranulocyte Influences adaptive immune Leukocyte responses + exert tissue repair function Macrophages Agranulocyte Phagocytosis and immune surveillance + inflammation Pathogen recognition and presentation Dendritic cells Agranulocyte Collect antigen and release Reside in superficial tissues cytokines that notify Connection between innate leukocytes and adaptive immunity Antigen presenting cells 5  BBS3014 – Małgorzata Maciążek Substances released by innate immune cells bactericidal substances → epithelial cells (skin, intestine, lungs, urogenital, salivary glands, tear glands etc) and immune cells (monocytes, neutrophils) lysozyme - enzyme that degrades peptidoglycan in tears, saliva, blood and immune cells peroxidases - enzymes that break down hydrogen peroxide to produce oxidizing compounds in the saliva and immune cells lactoferrin - high affinity for iron (needed for microorganisms to proliferate, so it limits proliferation and ability to enter the body in saliva, phagocytes, blood and tissue fluids defensins - antimicrobial peptides that form pores cytokines - mediators of communication between immune cells; used for communication between immune cells and other cell types bind to surface receptors and induce changes in cells: growth, differentiation, movement and cell death → interleukins → various functions, important in innate and adaptive immune system → chemokines → promote migration of cells to appropriate site → interferons - control of viral infections - promote tumour cell recognition local or systemic effects: promote inflammation (local) → increased permeability of endothelial cells + more adhesion molecules (IL-1, TNF) leukocytes release more of cytokines under influence of other cytokines cytokines affect hypothalamic centre in the brain = fever → decrease viral and bacterial replication and increase immune response acute phase proteins - biomarker of inflammation, cytokines force liver to make more complement proteins cytokines cause higher production of leukocytes chemotaxis - where the cells should go – stimulated by bacterial products, chemokines: monocyte chemo-attractant protein 1 - Mcp-1 → made by epithelial cells IL-8 → neutrophils use it to get to the site of infection a lot of complement activation - C3a and C5a → create a gradient 6  BBS3014 – Małgorzata Maciążek Physiological barriers of innate immune system breaking the barrier → grants entrance to tissues, blood and lymph → potential spread of pathogens (prevented by second line of defence = innate immune cells) First line of defence – mucosal barriers (urogenital tract, respiratory tract, eye, digestive tract and mucous barriers) Mucous barriers: → glycoproteins produced by goblet cells → sticky – taps microbes → propel pathogens to areas where they can be eliminated from Epithelial barrier of the skin → most difficult to penetrate (supported by keratin + a lot of layers of cells) → shedding of the outer layer which takes microbes with them → antimicrobial protein production (sebocytes and eccrine glands) Epithelial barrier of intestine → only one cell layer → tight junctions (JAMs and Zos) → goblet cells colon – thick very loose layer of mucosal cells 7  BBS3014 – Małgorzata Maciążek Development process of the immune cells: → innate immunity → active since birth, provides immediate, non-specific defence mechanisms → adaptive immunity → develops later, derived from hematopoietic stem cells (HSCs) in the bone marrow during fetal development → yolk sac and fetal liver (other pathway of resident immune cell development - immature DCs, mast cells and macrophages neonatal period → innate immune system relatively mature, adaptive immune system begins to develop (highest thymic activity + vaccine responses) Hematopoietic stem cells (HSCs): multipotent cells that are the origin of all immune cells (both myeloid and lymphoid) common myeloid progenitors (CMPs) common lymphoid progenitors (CLPs) Release of the immune cells from the bone marrow Immature cells in the bone marrow are retained by adhesive interactions with stromal cells and extracellular matrix. When cells mature → changes in adhesion molecules and chemokine receptors occur on cells → reduces adherence to bone marrow Chemokines such as SDF-1/CXCL12 and growth factors bind to receptors → direct the migration of mature cells towards blood vessels. 8  BBS3014 – Małgorzata Maciążek Exit bone morrow by moving through specialized blood vessels called sinusoids (wide, thin- walled capillaries) → cells interact with endothelial cells of sinusoids using adhesion molecules to adhere and migrate. Once passed through endothelial layer, cells enter blood stream. Negative feedback from circulating immune cells helps to regulate the production and release of cells, preventing overproduction or depletion. Migration of the immune cells Main function served by leukocyte migration from blood to tissue: 1. neutrophils and monocytes - arise in bone marrow, circulate in blood – later they are recruited into tissue sites of infection/injury, where they eliminate infectious pathogens, clear dead tissues and repair the damage 2. naïve lymphocytes – arise in bone marrow/thymus – they home to secondary lymphoid organs (lymph nodes, spleen), where they become activated by antigens and differentiate into effector lymphocytes 3. effector lymphocytes – develop in secondary lymphoid organs – they migrate into tissue sites of infection, where they participate in microbial defence → memory lymphocytes leukocyte homing (inflammation) → migration of leukocyte out of the blood to tissue or site of infection/injury recirculation → ability of lymphocytes to repeatedly home to secondary lymphoid organs, reside there transiently and return to blood When nothing happens: Naïve lymphocytes continuously migrate (secondary lymphoid organs) to ensure they are surveilling for antigens and can quickly respond if needed. Effector lymphocytes + myeloid lymphocytes home into tissues where there is infection/injury Memory lymphocytes migrate into lymphoid organs, mucosal tissues, skin and other tissues – distributed across various tissues to maintain state of being ready to respond to previously encountered antigens During an infection: Leukocyte rolling during migration towards injury and inflammation; initial contact with endothelial cells in post-capillary venules 1. Leukocyte rolling Macrophages, DCs → secrete cytokines (TNF, IL-1) which stimulate endothelial cells to express P-selecting and e-selectin → on activated endothelial cells (induced by pro-inflammatory cytokines TNF-α and IL-1, histamine, thrombin) 9  BBS3014 – Małgorzata Maciążek L-selectin → on leukocytes → their ligands are carbohydrates (PSLG-1) Selectins → interact with ligands on leukocytes, causing them to roll along endothelial surface due to low-affinity bonds → when blood is flowing it allows for rolling (attaches, pushed by blood, released and attaches again) 2. Activation of leukocytes Chemokine activation → displayed on the ES due to inflammation, presented by glycosaminoglycans (GAGs) Chemokines bind to chemokine receptors (g-coupled receptors) → causes intracellular signalling → activation of leukocytes/maturation of integrins → transitions from low-affinity to a high-affinity state 3. Firm adhesion high-affinity state → stronger more stable adhesive interaction with endothelium LFA-1 (lymphocyte function-associated antigen-1) binds to ICAM-1 (intercellular adhesion molecule-1) and ICAM-2 (endothelial cells) VLA-4 (very late antigen-4) binds to VCAM-1 (vascular cell adhesion molecule-1) VCAM-1 - infected cell; ICAM-1 - lymphoid organs those interactions cause the leukocyte to stop rolling and adhere to the endothelium 4. Crawling after firm adhesion → crawl for suitable location to transmigrate due to chemokine gradients Mac-1/ICAM-1 interactions→ promote lateral crawling, flattens the cell 5. Diapedesis leukocyte - loosen endothelial junctions by disruption of junctional adhesion molecules (JAMs) and VE-cadherin (vascular endothelial cadherin) PECAM-1 (platelet endothelial cell adhesion molecule-1; on both endothelium and leukocyte) facilitates this process through homophilic interactions → PECAM-1 pushed neutrophil flat in between gaps cytoskeletal reorganisation → allows endothelial cells to contract and create gaps for leukocytes to pass through + leukocytes can extend membrane protrusions to probe and penetrate the endothelial cell membrane 6. Basement membrane transmigration degradation of the basement membrane (dense network of extracellular matrix proteins like laminins, collagen, and fibronectin) done by matrix metalloproteinases (MMPs) + other proteases = clear path for migration chemokines + other signals guide leukocytes toward site of infection 10  BBS3014 – Małgorzata Maciążek Pattern recognition receptors pattern recognition receptors (PRRs) → critical component of innate immune system, responsible for detecting molecular patterns associated with pathogens/damaged cells they recognise pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) PAMPs → structure on the microbe (bacteria, viruses, fungi) → LPS, dsRNA, ssRNA DAMPs → released from stressed, injured, dying cells → ATP when outside of the mitochondria, crystals produced, HSP proteins (stress-induced) once detected → initiate immune response aimed at clearing the infection/repairing tissue damage Types of PRRs: 1. Toll-like receptors (TLRs) best-known, key role in recognizing a wide variety of pathogens, on: macrophages, dendritic cells, B cells, T cells, mast cells (histamine) and epithelial cells transmembrane, located on: cell surface (TLR1, TLR2, TL4, TLR5, TLR6) → recognise bacterial and fungal components within endosomal membranes (TLR3, TLR7, TLR8, TLR9) → recognise viral nucleic acids structure: extracellular domain with leucine-rich repeats (LRRs) for ligand recognition and an intercellular TIR domain (toll/IL-1 receptor domain for recognition 11  BBS3014 – Małgorzata Maciążek receptor ligands location pathway function TLR1 Lipoteichoic acid (gram Extracellular NF-κB inflammatory positive bacteria) TLR2 Lipoteichoic acid (gram Extracellular NF-κB + IRF3 Inflammatory, positive bacteria) and antiviral zymosan (fungi) TLR3 dsRNA (virus) Intracellular NF-κB + IRF3 Inflammatory, antiviral TLR4 Lipopolysaccharide Extracellular NF-κB + IRF3 Inflammatory, (LPS) (gram-negative antiviral bacteria), HPS proteins TL, R5 Flagellin (motility of Extracellular NF-κB inflammatory bacteria) TLR6 Lipoteichoic acid (gram- Extracellular NF-κB inflammatory positive bacteria) TLR7/8 ssRNA, ssDNA from Intracellular IRF7 antiviral viruses TLR9 Unmethylated CpG DNA Intracellular IRF7 antiviral (bacteria, viruses, and fungi) Signalling pathways explained: extracellular → gene expression → inflammatory function ligand binding → MyD88 → (transcription factors) NF-κB, I → (pro-inflammatory cytokines) TNF-α, IL-6, IL-1β intracellular receptors → antiviral pathway ligand binding → TRIF → (transcription factors) RF3, IRF7 → (interferons) IFN-α, IFN-β what does interferon do? → neighbouring cells that are still healthy are being put in antiviral state (prepares for infection of yet healthy cells) 2. C-type lectin receptors (CLRs) ligands: recognise carbohydrates structures on pathogens (mannose, glucans and fucose); mostly: membrane-bound collectin, ficolens - complement innate immune, mannose-like receptors, they promote phagocytosis (passive function) mediates phagocytosis, initiates immune signalling and induces cytokine production (IL-6, IL- 23) → supports Th17 responses crucial for fungal immunity, on: macrophages (mannose) and dendritic cells (dectin-1) 12  BBS3014 – Małgorzata Maciążek receptor ligands Dectin-1 β-glucans (fungal cell walls) Mannose receptor mannose and fucose (microbial surface) Signalling pathway: Syk kinase/CARD9 → (transcription factors) NF-κB, AP-1 → (pro-inflammatory cytokines) + immune cell recruitment 3. NOD-like receptor (NLRs) cytoplasmic receptors sense intracellular PAMPs and DAMPs - vital role in inflammation and immune homeostasis they detect microbial components (bacterial cell wall fragments (peptidoglycans) and bacterial toxins, stress signals) located on: (mucosal) endothelial cells, macrophages (tuberculosis), dendritic cells, not neutrophils! receptor ligands NOD1 DAP (γ-D-glutamyl-meso-diaminopimelic acid) in gram negative, bacterial proteins and viral RNA NOD2 muramyl dipeptide (MDP) in gram-positive and gram-negative bacteria, bacterial proteins and viral RNA NLRs → NF-κB or induce inflammasome formation, which leads to pyroptosis (a form of inflammatory cell death) + release of pro-inflammatory cytokines NOD2 - type I interferon NLRA and NLRB NOD1 and NOD2 - NLRC subfamily NLRP3 (NLRP subfamily) → form inflammasomes - multiprotein complexes that activate caspase-1, leading to the maturation of IL-1β and IL-18, potent inflammatory cytokines 4. RIG-I-like receptors (RLRs) cytoplasmic receptors, detect viral RNA within infected cells, triggering antiviral responses found on: epithelial cells, bone marrow derived leukocytes, various tissue types receptor ligands RIG-I ssRNA, dsRNA (viruses) MDA5 Long dsRNA (viruses, picoviruses) 13  BBS3014 – Małgorzata Maciążek signalling pathway: RLRs → signalling pathway → IRF3, IRF7 → type I interferons (IFN-α and IFN-β) RLRs → NF-κB → pro-inflammatory cytokines (through MAVS) 5. Scavengers’ receptors on: macrophages → extracellular, on cell surface help with phagocytosis receptor ligands CD36 LPS, lipoteichoic acid, nucleic acids, β- glucans SR-A LPS, lipoteichoic acid, nucleic acids, β- glucans 6. Cytosolic DNA sensors on cytosol on many different cells double-stranded DNA (dsDNA) from pathogens or damaged cells (host cells too) 1→ STING pathway → induces type I interferons (antiviral response) + enhances autophagy (degrading of organelles) 2→ STING independent → two types: RNA polymerase 3 → RIG-1 pathway → type I IFN expression AIM2-like receptors → inflammasome → caspase-1 activation + promotion of IL-1β and IL- 18 release 14  BBS3014 – Małgorzata Maciążek Neutrophils most abundant and principal cell type in acute inflammatory responses circulate as spherical cells with membranous projections - nucleus segmented in 3-5 connected lobules connected by strands of chromatin → polymorphonuclear leukocytes (PMNs) 2 membrane-bound granules - filled with enzymes (lysozyme, collagenase and elastase) survive between few hours up to 5 days (half-life of 6-12 hours) → after entering tissues, neutrophils function for only 1 to 2 days and most of them then die Development: produced in bone marrow, stimulated by granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) Myeloblast → Promyelocyte → Myelocyte → Metamyelocyte → Band Cell → Mature Neutrophil Functions: phagocytose microbes, especially opsonized microbes, and products of necrotic cells and destroy these in phagolysosomes degranulation release the contents of their granules into the extracellular environment during infections, they degrade microbial membranes and help neutralise pathogens neutrophil extracellular traps - NETs produced by neutrophils after pathogen recognition→ helps to limit the spread of infection but can contribute to tissue damage and autoimmune diseases if dysregulated NETosis → they release their DNA contents, histones and granules, which forms a mesh network with high levels of the antimicrobial proteins, which captures the bacteria and kills them (also neutrophils die) 15  BBS3014 – Małgorzata Maciążek release of cytokines and chemokines for recruitment of new cells reactive oxygen species (ROS) production production of large amounts of ROS through the NADPH oxidase complex during respiratory burst trigger: engulfing a pathogen mediated by NADPH oxidase → this complex generates ROS toll like receptor → NF-κB → phagocyte oxidase 1. activation of NADPH oxidase when pathogen is phagocytosed →phagocytotic cell activates its NADPH oxidase enzyme complex (on the membrane of the phagosome) NADPH oxidase transfers electros from NADPH to molecular oxygen, converting it into superoxide (within phagosome) NADPH → (NADPH oxidase) → O₂ → O₂⁻ 2. formation of ROS superoxide dismutase into hydrogen peroxide, under superoxide dismutase (SOD) hydrogen peroxide reacts with chloride ions, catalysed by myeloperoxidase (MPO) (on neutrophil granules) to produce hypochlorous acid other ROS: hydroxyl radicals (OH ), singlet oxygen (O₂), and nitric oxide (NO) O₂⁻ → (SOD) → H₂O₂ → H₂O₂+Cl⁻ → HOCl HOCl - extremely potent antimicrobial agent ROS: function: Superoxide (O₂⁻) very reactive, can directly damage microbial proteins Hydrogen Peroxide (H₂O₂) less reactive, precursor for others Hypochlorous Acid (HOCl) powerful oxidizing agent, destroys a wide range of pathogens by causing oxidative damage to microbial proteins, lipids and nucleic acids Hydroxyl Radical (OH ) most reactive and destructive, can damage proteins, lipids and DNA = death of the microbe Ways to killing via oxidative burst: lipid peroxidation → damage microbial membranes, making them leaky and dysfunctional protein oxidation → modify and inactive essential microbial enzymes and structural problems DNA damage → can cause strand breaks and mutations in the pathogen’s DNA = death 16  BBS3014 – Małgorzata Maciążek Regulation by: enzymatic scavengers (catalase, glutathione peroxidase, superoxide dismutase) → helps neutralize excess ROS and protect phagocytic cell itself from oxidative damage apoptosis and clearance → neutrophils undergo apoptosis and macrophages clear apoptotic cells, minimizing the risk of excessive tissue damage from prolonged ROS exposure 17  BBS3014 – Małgorzata Maciążek Macrophages versatile immune cells that play a central role in body’s defence mechanisms, tissue homeostasis and inflammation Development: yolk sac derived macrophages → develop early in embryogenesis and remain in tissues throughout life (microglia in the brain, Kupffer cells in liver, alveolar macrophages and osteoclasts) monocyte-derived macrophages → monocytes from bone marrow, differentiate into macrophages based on local cues lifespan → long, months to years, they can multiply an exhibit plasticity → can adopt different phenotypes depending on the signals; 2 types based on polarization: M1 (classically activated macrophages) induced by pro-inflammatory signals (IFN-γ, LPS and TNF-α) involved in host defence against intracellular pathogens and cancer → produce high levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-12), generate reactive oxygen species (ROS) and nitrogen species + participate in tissue destruction to control infections marker expression: iNOS, MHC class II and CD86 (B7 molecule) M2 (alternatively activated macrophages) induced by anti-inflammatory signals (IL-4, IL-13, IL-10 and glucocorticoids) involved in tissue repair, wound healing and resolution of inflammation → produce anti- inflammatory cytokines (IL-10, TGF-β) + promote tissue remodelling, angiogenesis and debris clearance maintenance of tissue homeostasis subtypes: M2a - involved in tissue repair and wound healing M2b – immunoregulation M2c - tissue remodelling and anti-inflammatory responses marker expression: arginase- 1, CD163, CD206 (mannose receptor) and TGF-β 18  BBS3014 – Małgorzata Maciążek Functions: 1. Phagocytosis recognition: recognise targets via receptors (scavenger receptors, Fc receptor - for antibody- opsonized pathogens, complement receptors - for complement-opsonized pathogens, and PRRs) engulfment: engulfed into a phagosome, which fuses with lysosomes to phagolysosome, where hydrolytic enzymes degrade a pathogen (lysozyme, acid hydrolases) killing: enzymatic degradation + oxidative bursts, producing ROS and RNS to kill engulfed pathogens macrophage phagocytosis of healthy host cells → prevented by an inhibitory receptor on the macrophage SIRPα, which recognizes CD47, a membrane protein on healthy cells that functions as a “don’t eat me” signal when CD47 binds to SIRPα, → inhibitory signals are generated = prevention of phagocytosis low pH, enzymes like: NADPH oxidase, will still electrons from oxygen, which creates superoxide superoxide + super oxide dismutase -> forming of hydrogen peroxide hydrogen peroxide + chlorine ions and iron ions -> hydroxyl radical, hypochlorite ROS steals electrons from bacteria, which will kill them fast If no oxygen - alternative, only in macrophages Inducible nitric oxide synthase, it converts arginine into nitric oxide, which forms radicals that kill the microbes with other enzymes present in lysosomes TLR + IFN-γ → iNOS 19  BBS3014 – Małgorzata Maciążek iNOS catalyses arginine → citrulline = release of NO NO + hydrogen peroxide or superoxide → highly reactive per-oxynitrate radicals that can kill microbes NO → attacks what has been phagocytosed 2. Antigen presentation APCs (antigen presenting cells) - on MHC class II molecules present antigens to T cells, bridging innate and adaptive immunity after phagocytosing a pathogen, macrophages degrade it and present peptides to CD4+ T cells → T cell activation + involvement of adaptive immune response 3. Cytokine production → pro-inflammatory - TNF-α, IL-1β, IL-6, and IL-12; initiating and amplifying inflammation, recruiting other immune cells and promoting T helper 1 (Th1) responses → anti-inflammatory cytokines - IL-10 and TGF-β; produced to resolve inflammation, limit tissue damage and promote tissue repair → chemokines - CCL2 (MCP-1) and CXCL8 (IL-8); attract immune cells to sites of infection or injury 4. Wound healing and tissue remodelling clear apoptotic cells and debris - efferocytosis (clearance of dead cells) 20  BBS3014 – Małgorzata Maciążek secrete growth factors (VEGF for angiogenesis), produce matrix metalloproteinases (MMPs) to degrade and remodel extracellular matrix (ECM) 5. Role in inflammation and resolution pro-inflammatory → M1 promote inflammation by secreting cytokines and mediators sentinel cells - sense the presence of microbes and respond accordingly anti-inflammatory → M2 secrete cytokines, promote tissue repair and help clear remaining debris (inflammatory → reparative) efferocytosis → clearance by macrophages of apoptotic cells, including apoptotic neutrophils 6. Inflammasome activation cholesterol crystals, ATP levels, urea, efflux of potassium, cytosolic flagellin, urea → can lead to activation of inflammasome 3 parts: NLRP3 + adaptor protein + caspase-1 PRRs recognise PAMPs or DAMPs → activation and assembly of inflammasome (from NLRP3) → activation of caspase-1 → maturation of pro-inflammatory cytokines IL-1β (cleaves Pro-IL-1β into its active form) and IL-18⁠ ⇒ strong pro-inflammatory response⁠ 21  BBS3014 – Małgorzata Maciążek Complement system consists of several plasma proteins that work together to opsonize microbes, to promote recruitment of phagocytes to the site of infection and possibly directly kill the microbes activation → proteolytic cascades in which an inactive protein (zymogen) gets altered to become an active protease that cleaves and thereby induces the proteolytic activity of the next complement protein in the cascade enzymatic cascades = amplification of the number of proteolytic products that are generated at each step they perform the effector functions of the complement system 1. classical pathway one of the major effector mechanisms of the humoral adaptive immunity C1q detects antibodies (IgM, IgG) bound to the surface of microbes and binds to Fc portion of antibodies C1r + C1s become active + initiate proteolytic cascade Antibodies are first produced, complement molecule binds to C1 and then further bind to the antibody Which activates C4/C2 → C4b/C2b Which further activates C3 → C3a and C3b 22  BBS3014 – Małgorzata Maciążek 2. alternative pathway C3 spontaneous hydrolysis: when C3 (complement protein) recognizes LPS → spontaneously happening, continuously going on except of the inhibitory proteins that stop it from amplifying pathway can distinguish normal self from foreign microbes on the basis of the presence or absence of the regulatory proteins pathogen does not have inhibitory proteins - activation C3 → C3b, C3b binds to the pathogen Bf → Bb (under influence of factor D), Bb further binds C3b to the pathogen ^amplification loop with activation from pathogen 23  BBS3014 – Małgorzata Maciążek 3. lectin pathway triggered by mannose-binding lectin (MBL) which recognizes terminal mannose residues on microbial glycoproteins and glycolipids MBL is a member of the collectin family → hexametric structure similar to C1q after binding → MASP1 (mannose-associated serine protease 1 or mannan-binding lectin- associated serine protease) and MASP2 MBL (mannose binding lectin) recognized mannose which is only present on bacteria, and it’s bound by MBL MASP-1 and MASP-2 activate C4/C2 → C4b/C2b Which further activates C3 → C3a and C3b C3 convertase, cleaves the central protein of the complement system, C3, producing C3a and C3b C3b - covalently bound to microbial surface → amplification and C3b acts as opsonin to promote phagocytosis C3a - released and stimulates inflammation (chemoattractant for neutrophils, by inducing mast cell degranulation, and by directly increasing vascular permeability → plasma proteins and fluid leak into sites of infection) C3 + complement proteins = C5 convertase → cleaves C5 into C5a and C5b (attached to microbial cell membrane) C5a - proinflammatory effects C5b - formation of a complex of the complement proteins C6, C7, C8, and C9 into MAC (membrane attack complex) that causes lysis of cells where complement is activated 24  BBS3014 – Małgorzata Maciążek Regulation: alternative pathway → C3 - activated at a low level in the blood and extravascular fluid and binds to cell surfaces → inhibited by regulatory molecules present on mammalian cells → microbes lack these regulatory molecules, therefore spontaneous activation can be amplified on microbial surfaces (self-tolerance) Summary: 25  BBS3014 – Małgorzata Maciążek Innate lymphoid cells LCs → a group of immune cells that play a critical role in both innate immunity and tissue homeostasis; resemble T cells but no antigen-specific receptors mostly in the tissues, they take shorter time to activate and react than T helper cells → but can stimulate further immune response important in early immune repones to infections, inflammation and maintaining tissue integrity at mucosal surfaces (gut, lungs, skin) divided into 3 groups based on cytokine production and transcription factor requirements: Type: Production: Function: Example: ILC1 IFN-γ (similar to Th1) defence against NK cells intracellular pathogens (viruses and bacteria) ILC2 Il-5, IL-13, and IL-9 defence against (similar to Th2) parasitic infections (helminths) and allergic reactions (asthma) ILC3 Il-17 and Il-22 (similar defence against lymphoid tissue Th17) extracellular bacteria inducer (LTi) cells - and fungi (especially essential in in gut) organogenesis Function: 1. barrier immunity - maintaining homeostasis at mucosal barriers, where they coordinate early responses to pathogens, regulate microbiota and promote tissue repair 2. pathogen defence ILC1/NK cells → defence against intracellular pathogens (virus, bacteria) and produces IFN-γ to activate macrophages and promote cytotoxic activity ILC2 → responds to parasitic infections and allergens, contributes to type 2 immunity by secreting IL-5 and IL-13 ILC3 → defend extracellular bacteria and fungi through production of IL-7 and IL-22, which maintains epithelial integrity and stimulate production of antimicrobial peptides 3. tissue homeostasis and repair ILC2 → contribute to wound healing in lungs and gut by secreting amphiregulin → promotes epithelial cell proliferation and repair ILC3 → maintain intestinal homeostasis by regulating the balance between tolerance to commensal bacteria and defence against pathogenic organisms 26  BBS3014 – Małgorzata Maciążek 4. inflammation and disease overactive ILCs contribute to allergic diseases (asthma, atopic dermatitis and chronic rhinosinusitis ILC3s are linked to inflammatory bowel disease (IBD) → Crohn’s disease and ulcerative colitis Development: → fatal liver + primarily in bone marrow from CLPs (common lymphoid progenitors) but they branch off early due to its lack of recombination-activating gene (RAG)-dependent receptor rearrangement → proceed down a developmental pathway driven by specific transcription factors early innate lymphoid progenitor (EILP) - key intermediate that retains the capacity to differentiate into all ILCs but not adaptive lymphocytes common helper-like innate lymphoid progenitor (CHILP) - LTia, gives rise to ILC2 and ILC3 but not NK cells or T cells which transcription factors are needed for each cell type: ILC1/NK cells → T-bet and Eomes (eomesodermin) ILC2 → GATA3 and Rorα ILC3 → RORγt and AHR (aryl hydrocarbon receptor) microenvironmental signals → shaped by tissue- specific cues (cytokines, growth factors and microbial products) ILC2 → IL-7 and IL-33 ILC3 → IL-1β, IL-23, and IL-7 maintenance and activation: rare and maintained in tissues in a quiescent state under normal conditions if infection or tissue damage → local cues (DAMPs or cytokines: IL-25, IL-33) activate ILCs, which causes proliferation and secrete effector cytokines that shape the immune response Activation: ILCs are in mucosal barriers (gut, lungs, skin) → exposed to cytokines from epithelial and stromal cells, and resident immune cells in response to infections or tissue damage 27  BBS3014 – Małgorzata Maciążek alarmins: IL-25, Il-33 and TSLP (thymic stromal lymphopoietin), released during tissue damage or pathogen invasion = activation indirectly activated by DAMPs and PAMPs → not by PRRs, other immune cells recognise them and release cytokines Type of the cell: Key activating signals: Produced cytokines: ILC1 IL-12, IL-18 and IL-15, stress IFN-γ, TNF ligands (e.g., MIC-A/MIC-B for NK cells) ILC2 IL-25, IL-33, TSLP, IL-4 IL-5, IL-13 and IL-9 ILC3 IL-1β, IL-23, IL-7 IL-17, IL-22 Regulatory inputs + modulators of ILC activation: 1. tissue-specific cues lung: ILC2 are primed to respond to allergens and helminths gut: ILC3 are more responsive to bacterial signals 2. metabolic and microbiota signals short-chain fatty acids (SCFAs) produced by commensal microbes - modulate ILC activation (ILC3) by influencing cytokine production and barrier function 3. neuro-immune interactions neuronal signals (VIP - vasoactive intestinal peptide; neuromedin U) can directly activate ILCs (ILC2) influencing response during allergic reactions and tissue repair processes plasticity → ILCs can switch from one type to another under certain conditions ILC3-to-ILC1 plasticity → in response to IL-12, which has been associated with intestinal inflammation allows for functional flexibility in response to environmental changes/disease 28 BBS3014 – Małgorzata Maciążek 29  BBS3014 – Małgorzata Maciążek NK cells type of ILCs, critical role in the immune systems defence against virally infected cells and tumours; do not require prior sensitization or antigen-specific receptors to recognise and eliminate target cells rapid response to “stressed” cells that exhibit changes (infection, malignant transformation, damage) by killing these cells directly and secreting cytokines to modulate the broader immune response they have different types of killing → granzymes is the fastest way of killing CD56 level is different in 2 subtypes of NK cells: bright CD56 - killing NK cells dim CD56 - cytokine producing they are helper NK cells, can be found in low levels in the blood → by producing more cytokines - they become more anti-inflammatory, with more of a regulatory function can become memory cells in the blood → most mature state - with Cd56 dim expression (killers) → pro-inflammatory function/cytotoxic function 30  BBS3014 – Małgorzata Maciążek Functions: 1. cytotoxicity (fast) direct killing → induce apoptosis in target cells via release of cytotoxic granules: perforin → creates pores in target cell membrane granzymes → (serine proteases) enter the target cell through pores and trigger apoptosis by activating caspases (slow) death receptor ligand interactions → receptor-ligand interaction causes caspase activation (pro-apoptotic pathways) NK cells express ligands like FasL (Fas ligand) and TRAIL (TNF-related apoptosis-inducing ligand), which binds to death receptor Fas (CD95) and TRAIL-R on target cells, which leads to apoptosis through caspase activation 2. antibody-dependent cellular cytotoxicity (ADCC) NK cells mediate ADCC through CD16 (FcγRIIIa), which recognizes IgG antibodies bound to surface of a target cell → triggers the release of cytotoxic granules, leading to the killing of target cell 3. cytokine production NK cells secrete IFN-γ, TNF-α, and GM-CSF that modulates immune responses - they activate macrophages and DCs, enhance antigen presentation and stimulate adaptive immune responses IFN-γ - activates macrophages to enhance phagocytosis and pathogen killing, promotes Th1 polarization of CD4+ T cells and upregulates MHC class I expression on target cells, which makes them more recognizable to cytotoxic T cells macrophages as a host for intracellular bacteria - tuberculosis → he is escaping from regular phagocytosis process inside the bacteria how to deal with it? - NK cell makes IFN-γ and binds through it, which causes overproduction of cytotoxic cytokines Development: from CLPs (common lymphoid progenitor cells) in the bone marrow, sharing a lineage with T cells and other ILCs Development is driven by: IL-15 → essential for NK cell development, proliferation and survival IL-2 and IL-12 → maturation and activation of NK cells transcription factors: eomesodermin (Eomes) and T-bet → regulate NK cell lineage commitment and functional specialization once mature → circulation in the blood + present in peripheral tissues (spleen, liver, lungs and lymphoid organs) 31  BBS3014 – Małgorzata Maciążek pro-NK cells (stage 1) → pre-NK cells (stage 2) → immature NK cells (stage 3) → Cd56 bright (stage 4) → CD56 dim (stage 5) Activation: activated through a balance between activating and inhibitory receptors on their surface → they recognize range of stress signals and changes in the surface of target cells → very redundant system - if we take one of the receptors out, we don’t know if we can get the same type of activation/inhibition anymore 1. inhibitory receptors NK cells are prevented from attacking healthy cells by inhibitory receptors that recognize self-molecules - major histocompatibility complex (MHC) class I molecules (on the healthy cell in question) interaction by MHC I and inhibitory receptor - cell is recognized as “self” and protected from killing killer-cell immunoglobulin-like receptors (KIRs) → bind to specific MHC class I molecules (HLA-A, HLA-B, HLA-C) CD94/NKG2A → recognizes the non-classical MHC class I molecule (HLA-E) they send inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains, which recruits phosphates (SHP-1, SHP-2) to dampen activation signals 2. activating receptors NK cells get activated when they sense stress-induced ligands or when MHC class I molecules are downregulated (a hallmark of viral infection or tumour transformation) ⇒ triggers NK cell activation 32  BBS3014 – Małgorzata Maciążek NKG2D - recognizes stress-induced ligands (MICA, MICB, ULBP) that are upregulated on infected, stressed or transformed cells Natural cytotoxicity receptors (NCRs) NKp30, NKp44, NKp46 → bind various stress ligands in tumour cells or virally infected cells and play a crucial role in target cell recognition and cytotoxicity CD16 (FcγRIIIa) - mediates antibody-dependent cellular cytotoxicity (ADCC) by binding to Fc portion of IgG antibodies coating a target cell, triggering NK cell activation they send signals through immunoreceptor tyrosine-based activation motifs (ITAMs), which initiate intracellular signalling cascades involving kinases (Syk, ZAP-70) leading to NK cell activation, degranulation and cytokine production → inhibitory signal will be stronger than the activation signal if both receptors are bound 3. Cytokines 4. Antibodies → one of the strongest NK responses 5. virus-derived ligand → viral proteins NK cell - heathy cell → tolerance/acceptance (”missing self”) NK cell - tumour cell (no more HLA/MHC-I + needs a little bit of stress ligand) → activation no MHC-I reduces the activation threshold (”induced self”) NK cell - tumour cell (they still have MHC-I, a lot of activating signals) → activation 33  BBS3014 – Małgorzata Maciążek NK cell education/NK cell licencing → controls responsive potential of the cells to avoid hyper- or hypo-activation (similar to T cell “teaching” in the thymus) we do not know where it actually happens learning: interaction between inhibitory receptor that bind to HLA, which matures NK cell if it does not happen → the NK cells stay around ad unlicensed (less activated, less granules, less activating receptor, which makes them not as good responder) What are adaptive features of NK cells? Virus-induced memory NK cells Memory not as good as B cells → they also have activating receptors with HLA/MHC-I In some cases, the same ligand that inhibits NK cells can activate NK cells → it is not black and white (NKG2 and KIR) KIR has a lot of polymorphisms → they are inherited through generations that's why people might be suspectable to certain types of cancer 34  BBS3014 – Małgorzata Maciążek can you use NK cells from one donor into a different patient? → you can, the receptors are the same in all of the people (we all have MHC-I the same way) immunological synapse - NK cell and target cell have to make contact through a receptor, but you have to create an attachment between each other → they have adhesion molecules and create a bridge → in between - released enzymes to kill NKG2C+ → activating receptor that recognised the viral proteins in MHC-I → proliferation and epigenetic modification to make the stronger NK cells (memory NK cells → they have much longer life span) 35  BBS3014 – Małgorzata Maciążek if you re-activate the memory NK cells → production of interferon gamma (IFN-γ) cytokine-induced memory cells if you have right cytokine cocktails → proliferation and metabolism modifications, which creates better effector cells that stay in the body longer (”long-lived”) Upregulation of IFN-γ, CD25, perforin and granzymes NK cell homing and trafficking → liver, lung (Th1 function), uterine decidua (attract foetus blood vessels, arteries remodelling) and lymph nodes 36  BBS3014 – Małgorzata Maciążek Dendritic cells tissue-resident and circulating cells that detect presence of microbes and initiate innate immune defence reactions; they capture microbial proteins for display of T cells to initiate adaptive immune responses (APCs - antigen-presenting cell) named because of their long membranous projections - similar to neurons distributed in lymphoid tissues, mucosal epithelium and organ parenchyma thanks to that they capture antigens and take them to lymph nodes where naive T cells circulate and help to activate them sentinels of immune system → they initiate innate immune response and also tie innate and adaptive immune responses Types of dendritic cells: 1. classical/conventional dendritic cells (cDC) major types of DCs, involved in capturing protein antigens of microbes that enter through epithelial barriers and presenting them to T cells most numerous DC subset in epithelia and lymphoid organs cDC1 → specialize in cross-presentation of antigens to CD8+ T cells, critical for anti-viral and anti-tumour immunity cDC2 → involved in activating CD4+ T cells (helper T cells) and orchestrating a broader immune response (both Th1, Th2, and Th17 pathways) 2. plasmacytoid dendritic cells resemble plasma cells morphologically, main producers of cytokines - IFN I ability to produce large amounts of type I interferons during viral infections, thus playing a crucial role in antiviral immunity less efficient in antigen presentation compared to cDCs but contribute to activating immune responses through cytokine production 3. monocyte-derived dendritic cells (moDCs) can differentiate from monocytes in inflammatory conditions often generated in vitro for experimental purposes but play a significant role in inflammation and infection in vivo 4. Langerhans cells found in epidermis that share functions with cDCs but are developmentally related to tissue- resident macrophages (arise from embryonic fetal liver and yolk sac precursor) function in the context of skin infections to present antigens to and activate CD4 + T cells, or in the absence of infection, to present self-antigens to CD4 + T cells and induce tolerance to these antigens. 37  BBS3014 – Małgorzata Maciążek identified by their location and morphology in the skin, the presence of tennis-racket– shaped cytoplasmic organelles called Birbeck granules 5. follicular DCs - come back in the last case DCs morphology, not derived from bone marrow precursors, do not present protein antigens to T cells, involved in B cell activation in the germinal centres of secondary lymphoid organs have TLRs, NLRs, important for T-cell activation, MHC-I always (in healthy conditions - self antigen is expressed) 38  BBS3014 – Małgorzata Maciążek cDC2s cDC1s pDCs Langerhans moDCs cells Surface CD11c, CD11c, BDCA-2 CD11b CD11b, markers BCDA-1 BDCA-3 (CD303), Langerin CCR2, CD14 (CD1c) (CD141), BDCA-4 (CD207), CLEC9A, (CD304), EPCAM, XCR1+ CD123 BDCA1, CD1a TLRs various various various various expression Transcription IRF4 IRF8, BATF3 E2-2 PU.1 factors Cytokines various IL-12 type I IFN various produced Function source of early innate source of source of (innate) inflammatory response, inflammatory inflammatory cytokines priming of cytokines cytokines antiviral T cells Function capture and capture and antiviral capture and unknown (adaptive) presentation cross- immunity presentation of antigens presentation of antigens mostly to of antigens to mostly to CD4+ T cells CD8+ T cells; CD4+ T cells induction of Th1 responses 39  BBS3014 – Małgorzata Maciążek Development: from hematopoietic stem cells in the bone marrow - from both CMPs and CLPs: conventional DCs → from myeloid precursors, specialised in antigen presentation and T cell activation plasmacytoid DCs → from lymphoid precursors, key precursors of type I interferons in response to viral infections immature DCs: located in peripheral tissues (skin, mucosal surfaces) where they are highly specialized for antigen capture express PRRs to detect pathogens high phagocytotic activity → capturing pathogens, apoptotic cells or foreign particles and low expression of costimulatory molecules (CD80, CD86) necessary for T cell activation Antigen uptake: Immature DCs are highly efficient at capturing antigens, including pathogens, dead cells, and soluble molecules. This occurs through various mechanisms: Phagocytosis: Engulfment of larger particles like bacteria. Endocytosis: Internalization of smaller particles or molecules. Macropinocytosis: Non-specific uptake of extracellular fluid and soluble antigens. These antigens are processed into peptides, which can then be loaded onto MHC molecules for presentation to T cells. Maturation process upon exposure to pathogens, damage signals or inflammatory cytokines = maturation process DCs get activated by PAMPs and cytokines (TNF - tumour necrosis factor) and lose their adhesiveness for epithelia or tissues and begin to express a chemokine receptor called CCR7 → specific for two chemokines: CCL19 and CCL21, that are produced in lymphatic vessels and in the T cell zones of lymph nodes chemokines attract the DCs bearing microbial antigens into draining lymphatics and ultimately into the T cell zones of the regional lymph nodes naive T cells also express CCR7 → localize to the same regions of lymph nodes where antigen- bearing DCs are concentrated but via the blood characterised by: upregulation of surface MHC class I and II molecules to present antigens present self-antigens to self-reactive T cells and thereby cause inactivation or death of the T cells or generate regulatory T cells = important for maintaining self-tolerance (prevents autoimmunity) 40  BBS3014 – Małgorzata Maciążek upregulation of costimulatory molecules (CD80, CD86 and CD40) which are crucial for efficient T cell priming CD80 (B7-1) and CD86 (B7-2) bind to CD28 on T cells, providing the necessary "second signal" for T cell activation. Without this signal, T cells may become anergic (non- responsive). secretion of cytokines (IL-12, IL-6, IFN-alpha) which shape the adaptive immune response DCs lose their phagocytic ability and gain migratory properties by expressing chemokine receptors like CCR7, enabling them to migrate to lymph nodes. decrease the expression of adhesion molecules that keep them localised at the peripheral tissue ICAM3, OX40L, DC-sign antigens - transported to lymphoid organs in soluble form (B cells in the node may also recognize and internalize soluble antigens) resident DCs (from lymph nodes and spleen) may capture lymph- and blood-borne antigens OR may be driven to mature by microbial products → when lymph enters a lymph node through an afferent lymphatic vessel, it drains into the subcapsular sinus, and some of the lymph enters fibroblast reticular cell (FRC) conduits that originate from the sinus and traverse the cortex once in the conduits, low-molecular-weight antigens can be extracted by DCs (they line the outside surfaces of the conduits and whose processes interdigitate between the FRCs) other antigens in the subcapsular sinus are taken up by macrophages, which carry the antigens into follicles and present these antigens to resident B cells. mucosal surfaces of GI tract and respiratory system → contain specialized collections of secondary lymphoid tissue that can directly sample the luminal contents of these organs for the presence of antigenic material (Peyer’s patches of the ileum and the pharyngeal tonsils) mature DCs: → reside in the lymph nodes where they present processed antigen peptides bound to MHC molecules to naïve T cells Depending on the signals received during maturation, they promote differentiation of T cells into different subsets: CD8+ cytotoxic T cells: via cross-presentation of antigen with MHC class I. CD4+ helper T cells: via antigen presentation on MHC class II, with the ability to polarize T cells into Th1, Th2, Th17, or Treg cells based on the cytokines they secrete 41  ------ * 700/188 cytotoxic ⑫ class , MHC : HLA-A HLA. B HLAC , , class 2 MHC : HLA-DP HLA-DR HLA-DR ,. CLP RORyt ATA TE "El I ILF ILC2 Y ↓ 1217 1222 , 115 119 , 1413 IFNG , TNFa  BBS3014 – Małgorzata Maciążek MHC molecules Major histocompatibility complex (MHC) molecule - proteins found on the surface of cells that play crucial role in the immune system primary function:  to bind and present peptide antigens to T cells, enabling the immune system to recognize and respond to pathogens/infected cells/cancer cells  essential for distinguishing self from non-self type structure Function MHC class I α-chain → 3 extracellular present intracellular antigen domains, transmembrane to CD8+ cytotoxic T cell, region and cytoplasmic tail; which can kill infected or malignant cells - present on β2-microglobulin → non- most nucleated cell covalently associated protein that helps stabilize the structure; peptide binding cleft → α1 and α2 domains derived from intracellular proteins; α3 domain → interacts with CD8 co-receptors on cytotoxic T cells MHC class II α-chain and β-chain, each present extracellular antigens with two extracellular derived from pathogens or domains (α1, α2, β1, β2), a debris that has been engulfed transmembrane segment, and processed by APCs to and a cytoplasmic tail; CD4+ helper T cells peptide-binding groove - formed by α1 and β1 domains and is open ended allowing for longer peptides MHC class III located between MHC-I and encode immune-related MHC-II genes on the proteins such as chromosome complement proteins (C2, C4) and cytokines (TNF-α) 42  BBS3014 – Małgorzata Maciążek MHC genes diversity of MHC molecules - crucial for immune system to recognize and present a wide array of peptides from different pathogens encoded by genes located in the human leukocyte antigen (HLA) complex on chromosome 6 examples explanation MHC-I genes HLA-A, HLA-B, HLA-C highly polymorphic - there are many different alleles within population which contributes to genetic diversity MHC-II genes HLA-DP, HLA-DQ, HLA-DR significant polymorphism → HLA-DR has several alpha and beta chain genes allowing for various combinations characteristics:  most polymorphic genes in humans, meaning there are many different alleles in the population - allows different individuals to present and respond to a broad range of antigens  multiple MHC genes (HLA-A, HLA-B, HLA-C, etc.) are expressed in each individual → in haplotypes → inherited a set of MHC-I and MHC-II genes from both parents, providing a diverse set of antigen-presenting capabilities  maternal and paternal alleles of MHC genes are expressed, further enhancing the diversity of antigen presentation in an individual 43  BBS3014 – Małgorzata Maciążek  balancing selection - heterozygosity (having two different alleles at a given locus) → individuals can present a broader range of peptides, enhancing their ability to respond to diverse pathogens disease association:  HLA-B27 is associated with ankylosing spondylitis, an autoimmune disease affecting the spine.  HLA-DR4 is associated with rheumatoid arthritis, an autoimmune disorder.  HLA-DQ2 and HLA-DQ8 are linked to celiac disease, where the immune system reacts to gluten. Antigen presentation pathways process by which cells display antigenic peptides on MHC (Major Histocompatibility Complex) molecules for recognition by T cells 2 types of antigen presentation depending on the origin of the antigen: Endogenous pathway – MHC class I presenting intracellular antigens (viral or tumour-derived proteins) on MHC class I - active on all nucleated cells 1. protein synthesis and ubiquitination intracellular proteins are synthesized within the cell - proteins are tagged for degradation by ubiquitin 2. proteasomal degradation proteasome - large multi-protein complex, degrades the proteins into short peptide fragments (8-10 amino acids) - makes them suitable for MHC-I binding 3. peptide transport into the endoplasmic reticulum from cytosol → to the ER by TAP (transporter associated with antigen processing) complex 4. peptide loading onto MHC-I in the ER → MHC class I molecules assembly (α-chain and β2-microglobulin) peptides are loaded into the peptide-binding groove of MHC-I with the help of chaperone proteins like calnexin, tapasin, and ERp57 5. transport to the cell surface stabilization of the complex + transport via Golgi apparatus to the cell surface where it’s displayed for recognition by CD8+ cytotoxic T cells 44  BBS3014 – Małgorzata Maciążek Exogenous pathway – MHC class II presenting extracellular antigens (bacteria, fungi, debris from dead cells) on MHC class II molecules in professional antigen-presenting cell (APCs) like dendritic cells, macrophages and B cells 1. antigen uptake exogenous antigens are captured by APCs through phagocytosis, pinocytosis or receptor- mediated endocytosis captured antigens are contained within endosomes or phagosome 2. proteolytic processing endosome containing the antigen fuses with a lysosome = phagolysosome or endolysosome low pH and proteolytic enzymes (e.g., cathepsins) within the lysosome degrade the engulfed antigens into peptide fragments 3. MHC-II Synthesis and Transport MHC class II molecules (composed of α and β chains) - synthesized in the ER + assembled with a third component called the invariant chain (Ii) invariant chain blocks the peptide-binding groove of MHC-II, preventing premature binding of peptides within the ER 4. Invariant Chain Cleavage and Peptide Loading MHC-II-invariant chain complex is transported from the ER through the Golgi to a specialized compartment called the MHC class II compartment (MIIC) → invariant chain is cleaved, leaving CLIP (Class II-associated Invariant Chain Peptide**)** in the peptide-binding groove) HLA-DM, a specialized protein, facilitates the removal of CLIP and helps load the processed peptides onto the MHC-II molecule 5. transport to the cell surface MHC-II-peptide complex is then transported to the surface of the APC for presentation to CD4+ helper T cells 6. Migration to Lymphoid Organs: Chemokine Receptor Upregulation:  Mature DCs upregulate CCR7, a chemokine receptor that responds to CCL19 and CCL21, chemokines produced in the lymphoid organs. This enables the mature DCs to migrate from peripheral tissues to the T cell zones of secondary lymphoid organs (e.g., lymph nodes and spleen). Alteration in Motility:  Immature DCs are relatively sessile, but upon maturation, they acquire a migratory phenotype. This change allows them to travel through the lymphatic system to reach lymphoid tissues, where they can interact with T cells. 45  BBS3014 – Małgorzata Maciążek Cross presentation extracellular antigens on MH) , Cross-presentation refers to the process by which antigen-presenting cells (APCs)—especially dendritic cells (DCs)—present extracellular antigens on MHC class I molecules. CD141+ (BDCA-3+) DCs → very efficient, they have molecules specific for cross presentation (TAP1/TAP2 complex, specific proteases, and CD91, which plays a role in antigen uptake and processing) 1. antigen uptake DCs capture antigens through phagocytosis, receptor-mediated endocytosis, or macropinocytosis. antigens - derived from pathogens, dead cells, or soluble proteins 2. antigen processing - happens in cytosol or through vacuolar pathway cytosolic pathway → exported from endosomes into the cytosol, where they are degraded by the proteasome; resulting peptides are then transported into the endoplasmic reticulum (ER) or back into phagosomes via TAP (Transporter Associated with Antigen Processing) for loading onto MHC class I molecules vacuolar pathway → antigens are degraded within the endosomal/phagosomal compartments, and the resulting peptides are loaded directly onto MHC class I molecules within the vacuoles, bypassing the cytosol and TAP machinery 3. peptide-MHC class I complex formation - in the ER or in the endosomal compartments 4. T cell activation - antigen recognized by TCR of naive CD8+ T cells, leading to their activation, clonal expansion, and differentiation into cytotoxic T cells capable of targeting and killing infected or malignant cells 46  BBS3014 – Małgorzata Maciążek Adaptive immune system adaptive immunity (specific, acquired) - form of immunity that develops in a response to infection and adapts to it recognizes and reacts to antigens, it enhances protective mechanisms of innate immunity, making them more capable of combating microbes 2 major populations of lymphocytes: B lymphocytes and T lymphocytes features of adaptive immunity:  specificity and diversity (antigens recognised by determinants/epitopes; clonal selection - clones of lymphocytes with different specificities are able to recognize and respond to antigens)  memory (enhanced ability to respond to an antigen after previous exposure → faster, and stronger)  self-tolerance - does not react to own antigens  systematic - provides protection at distant sites  positive feedback loops amplify reaction + control mechanisms that prevent inappropriate reactions Humoral immunity Cell-mediated immunity Microbe Extracellular microbes phagocytosed microbes that can live within macrophages; intracellular microbes replicating in infected cells Responding lymphocytes B lymphocytes helper T lymphocyte or cytotoxic T lymphocyte Effector mechanism Secreted antibody cytokines, neutrophils and activated macrophage/killing infected cell Functions Antibodies prevent infections cytokine-activated and eliminate extracellular phagocytes kill microbes; microbes CTLs kill infected cells and eliminate reservoirs of infection 47  BBS3014 – Małgorzata Maciążek Cells of the adaptive immune system T cells (thymus cells) - recognise antigens (that have been prosses and presented in histocompatibility complex molecule) from CLPs, mature in the thymus (they undergo positive and negative selection)  killer T cells (CD8+) → kills infected cells with viruses, damaged or dysfunctional  helper T cells (CD4+) → regulate innate and adaptive immune responses, helps to decide which  regulatory T cells (Tregs) → maintain immune tolerance and prevent autoimmune responses B cells (bursa cells) - antibody molecule on the B cell surface which recognizes whole pathogens without any need for antigen processing from CLPs in the bone marrow, maturation in bone marrow and later in secondary lymphoid organs (spleen or lymph nodes)  plasma cells  memory cells 48  BBS3014 – Małgorzata Maciążek T cell development – T lymphopoiesis thymus becomes functional around 8th and 10th week of gestation → occurs primarily in the thymus development either in bone marrow or fetal liver from hematopoietic stem cells (HSCs) in the bone marrow → common lymphoid progenitor cells (CLPs) → differentiation into T cells specialised environment for T cell maturation, 2 main compartments:  cortex (differentiation happens here)  medulla (T cell selection processes) thymus epithelial cells (TECs), dendritic cells and macrophages → important in this process CLPs → precursor t cells → pro T cells → alpha/gamma T cells (from BM) or beta/delta T cells (from fetal liver) Key molecules Function Notch1 required for T cell linage to commit to the thymus IL-7 promotes survival and proliferation of thymocytes at different stages Pre-TCR signalling triggers beta selection during DN3 Double negative stages (DN) called negative stages because of absence of CD4 and CD8 surface markers T cell progenitors go through 4 stages (DN1-DN4) based on expression of surface markers (CD44, CD25): 1. DN1 (CD44+ CD25-) → immature thymocytes that are multipotent 2. DN2 (CD44+ CD25+) → cell begin to express the TCRβ (T cell receptor beta chain) = committed to T cell lineage 3. DN3 (CD44- CD25+) → β-chain rearrangement via VDJ recombination process, which is mediated by RAG1/2 if successful - cells begin to express pre-TCR complex (with an invariant pre-TCRα chain) 4. DN4 (CD44- CD25-) → cells proliferate and progress to the double-positive (DP) stage after beta selection Double positive stage (DS) cells express both CD4 and CD8 co-receptors and have completed beta chain rearrangement cells undergo TCR alpha chain recombination (through VJ recombination) to produce TCRαβ complex 49  BBS3014 – Małgorzata Maciążek TCRαβ complex undergoes positive selection → recognize self-MHC molecules presented by thymic epithelial cells (in cortex) to survive → more details below allelic exclusion → during development only one allele of the receptor is expressed (even though there are 2 copies of each gene - one from each parent)  allelic exclusion only for the beta chain (not for alpha chain)  TCR is produced from one set of gene segments, either maternal or paternal TCR → α and β chain, enzymes: RAG1/2 red blood vessel - where T cells come in, enter between medulla and cortex and migrate to outer layers (chemokine induced chemotaxis on connective tissue) delta Notch signalling → determines that progenitor becomes a T cells 1. β chain rearrangement → starts in T cell precursor V - variable, D - diversity, J - joining D-J joining → (DN1-DN2) V-DJ joining → (DN2-DN3) transcription and translation → into the TCRβ chain protein β + invariant (temporary) α chain → pre-TCR pairing 50  BBS3014 – Małgorzata Maciążek pre-TCR → sends a survival signal to the thymocyte thanks to which it progresses to next stage + later allelic exclusion checkpoint: if not correct → apoptosis 2. α chain rearrangement → later stage V-J joining → (DN3-DNP) once in DP → RAG1/2 gets disactivated transcription and translation → into the TCRα chain protein + no allelic exclusion (that’s why there can be multiple) pairing with β chain → complete TCRαβ ! no D segment in the α chain - simpler than the β ! zeta chain - important for intracellular signalling, works as an extension cord 51  BBS3014 – Małgorzata Maciążek Epigenetic regulation of gene transcription → environmental cue that opens up the DNA in order to get gene rearrangement it happens under influence of RAG1/2 → it created a loop that gets cut off and lost, it will never come back; the two ending are being connected into a coherent line Junctional diversity → filling in the gaps with P nucleotides (matching to where the gap was), which also means there is endless amount of TCR variants 52  BBS3014 – Małgorzata Maciążek 2 key selection processes → central tolerance: 1. Positive selection location: thymic cortex purpose → ensure T cells can recognize self-MHC molecules T cells that successfully bind to self-MHC class I → CD8+  IL-7 is important for CD8 → blocks the locus of one of CD4 T cells that successfully bind to self-MHC class II → CD4+(presentation by endosomes and merging, NOT cross presentation)  CD4 → they have inhibition of IL-7, which I think downregulates CD8 positive selection → downregulation of either CD4 or CD8 (just stop making one) → single positive cells cTECs - cortex thymic epithelial proteins - in MHC we have self-antigens → they cannot be empty (than they are degraded if they are) how is it regulated? → by chance depending on which one they encounter first, or they might have a preference (genetic encoded) → 2 hypothesis low-high affinity model → picture from the slide  if too low - die from neglect  if too high - die from auto-reactivity  if a bit lower than perfect → Tregs cells 53  BBS3014 – Małgorzata Maciążek 2. Negative selection location: thymic medulla → to eliminate T cells that recognize self-antigens too strongly (prevents autoimmunity) clonal deletion: cells that bind too strongly to self-peptides (tissue restricted antigens - TRAs, like insulin, myelin proteins, thyroid, liver, heart, spleen etc antigens thanks to AIRE) presented by medullary thymic epithelial cells (mTECs) = apoptosis the ones that do not bind → move on some T cells that recognise self-antigens with intermediate affinity → not deleted but changed into regulatory T cells (Tregs) they express: CD4, FOXP3 and CD25 (high-affinity IL-2 receptor) they: maintain immune tolerance in the thymus and periphery 54  BBS3014 – Małgorzata Maciążek M plasticity of thymic medullary epithelium:  single-cell heterogeneity → mTECs present self-antigens (mostly for periphery) via MHC to T cells, recognition = death  stereotypic stochasticity - some cells are expressed at the single moment but it’s very dynamic does not have to be the same  dynamics of ectopic gene expression  novel mode of gene activation thymic epithelial cells help with shaping the T cells → if less presentation a lot of autoreactive T cells → a lot of dying → more infections 55  BBS3014 – Małgorzata Maciążek Maturation from cortex to medulla red blood vessel - where T cells come in, enter between medulla and cortex and migrate to outer layers (chemokine induced chemotaxis on connective tissue) delta Notch signalling → determines that progenitor becomes a T cells here we have double negative stages DN1-3 later we become DP (double positive) - beta alpha locus is being made (full commitment) migration towards medulla where they interact with epithelial cells = positive and negative selection 56  BBS3014 – Małgorzata Maciążek AIRE (autoimmune regulator) - transcription factor expressed by mTECs that enables the expression of tissue specific antigens, which allows negative selection against a broad range of self-antigens multidomain structure, localizes to chromatin enclosing target genes, binds to histones and offers an anchorage to multimolecular complexes involved in initiation and post-initiation events of gene transcription translates almost all of the self-antigen proteins, exceptions (immune privileged places)  brain proteins are not expressed by the thymus sue to blood-brain barrier  eyes - not a lot of blood circulation  reproductive organs (testis and ovaries, placenta and fetus) mTECs make the self-antigen with AIRE and then dendritic cell can present it to the T cells 95% of the T cells die in the thymus 57  BBS3014 – Małgorzata Maciążek Lineage commitment and exit after surviving positive and negative selection - single-positive CD4+ and CD8+ = mature naive T cells exit the thymus and enter peripheral blood circulation and lymphoid tissues → remain in resting state until encountering specific antigen the ones which escape → anergy, Treg suppression and activation-induced cell death if you have autoreactive T cell: 1. anergy - functional inactivation when T cell encounters an antigen without co-stimulatory signals it becomes anergic (non- responsive → they do not proliferate or secrete cytokines ) B7 is lacking how it works: peripheral tissue often express self-antigens without providing these signals 2. Tregs suppression they secrete inhibitory cytokines that inhibit effector T cell activation (IL-10, TGF-β) direct cell-to-cell contact - inhibitory receptors (CTLA-4) can outcompete effector T cells for co- stimulatory molecules on APCs they can also deprive effector T cells of growth factors (IL-2) by absorbing them via CD25 → they have increased expression of the IL-2 receptor (CD25) 3. AICD - activation-induced cell death repeated activation in absence of infection/inflammation → mediated by Fas-FasL interactions or pro-apoptotic factors (Bim) Fas (CD95) - receptor on activated cell and interaction with a ligand = activation of apoptotic pathway 4. ignorance never encounter specific antigen in the periphery because antigens are focused in immune- privileged sites (brain, eyes, testes) where T cells do not have access to after differentiation process → potential autoimmunity if barrier breakdown occurs 5. co-stimulatory modulation - inhibitory signals co-inhibitory receptors:  CTLA-4 - cytotoxic T-lymphocyte-associated protein 4 competes with CD28 for binding to B7 molecules (CD80/CD86) on APCs - which delivers inhibitory signal that limits T cell activation  PD-1 - programmed death 1 if engaged by ligand (PD-L1, PD-L2) transmits an inhibitory signal that reduced T cell activity 58  BBS3014 – Małgorzata Maciążek T cells – all information cytokines produced by APCs or T cells → influence the differentiation into different subsets of effector cells:  CD4+ T cells → Th1, Th2, Th17 or T regulatory (Treg) cells  CD8+ T cells → effector T lymphocytes (CTLs) Types of T cells: Th-1 induced by:  IL-12 – produced by DCs and macrophages  IFN-γ – amplifies polarization through positive feedback loop  2 STAT molecules (STAT1 and STAT4)  STAT1 activate T-bet → gene transcription of IFN-γ