Pharmaceutical Immunology Summary PDF

Pharmaceutical Immunology Chapter 1 – Basic Concepts in Immunology The beginnings: Edward Jenner → vaccination = inoculation of healthy individuals with inactivated/attenuated pathogens or pathogenic constituents to induce protective immunity smallpox → eradication (1979) Main function of immune sy...

Pharmaceutical Immunology Chapter 1 – Basic Concepts in Immunology The beginnings: Edward Jenner → vaccination = inoculation of healthy individuals with inactivated/attenuated pathogens or pathogenic constituents to induce protective immunity smallpox → eradication (1979) Main function of immune system = protection from infection -> leukocytes (T cells, B cells, NK cells, eosinophil, basophil, neutrophil, immature dendritic cell, monocyte) & lymphoid organs (primary generate immune cells → bone marrow & thymus; secondary induce adaptive immune response → Peyer’s patches, spleen, tonsils, appendix, lymph nodes) Bone marrow: pluripotent hematopoietic stem cells give rise to leukocytes, erythrocytes, platelets by common lymphoid progenitor & common myeloid progenitor (all innate) Pathogen categories: - Viruses - Intracellular bacteria - Extracellular bacteria, archaea, protozoa - Fungi - Parasites ➔ Different in sizes ➔ Different places where reside Levels of defense: adaptive immunity takes long to establish, innate tries to keep pathogen in check Innate immune system Microbes have pathogen-associated molecular patterns (PAMPs) → detected by specific receptors on sensor cell in tissue/blood → produce inflammatory mediators (cytokines/chemokines), amplification by inducing antimicrobial/antiviral factors, recruitment & activation of other leukocytes Pattern recognition receptors: initial discrimination between self & nonself, for example Toll-like receptors (TLRs) on macrophages in skin Adaptive immune system During infection lymphocytes activated: - T cells: recognise & destroy infected cells, activate other leukocytes - B cells: activated by pathogen-specific T cells to secrete antibodies - Antibodies: bind specifically to foreign structure = antigens & make inactive Dendritic cells (DCs): major antigen- presenting cells in body, micropinocytosis, T cells antigen-specific ➔ Key link between innate & adaptive immune system Most T lymphocytes constantly recirculate between blood & lymph nodes (blood → lymph node → efferent lymphatic vessels → thoracic duct → blood) to increase chance of finding cognate antigen → find one on antigen- presenting DC: clonal selection: upon recognition of foreign antigen naïve lymphocyte activated → proliferates & differentiates → army of identical, antigen-specific T cells, acquire effector functions Antibody: Y-shaped, 150 kD, 2 heavy & 2 light chains, linked by disulfide bridges, also as transmembrane protein on original B cell Variable region: site of antigen binding, different amino acid sequence in different antibodies Constant region: identical in antibodies of same subtype, Fc part interacts with phagocytes & NK cells (Fc receptors) In plasma & extracellular fluids → humoral immunity  Epitope/antigenic determinant (small portion of antigen’s molecular structure recognised by antibody), antigen (protein, glycoprotein, polysaccharide of pathogen), self-antigen Neutralisation also prevents viruses from binding to receptors Antibody titer = concentration of antibody in blood (often decreases over time after vaccination) Difference Ab to TCR: Ab binds directly to native antigen, TCR only recognises peptide fragments of antigen presented on MHC molecules Antigen recognition by TCR: TCR: α and β chains linked by disulfide bridge Variable region: site of binding to peptide-MHC molecules, different amino acid sequence in different T cell clones Constant region: identical in most T cells Major histocompatibility (MHC) molecules: MHC class I MHC class II On all cells in body, present fragments of Expressed by antigen-presenting cells (APC: proteins expressed by cell itself, recognised by DCs, macrophages, B cells), present fragment of cytotoxic CD8+ T cells (& killed) proteins taken up into APC from outside (phagocytosis/micropinocytosis or B cell via B cell receptor), interact with CD4+ T cells Lymph node: Outermost cortex: B cell follicles, T cell zones Inner medulla: macrophages, antibody-secreting plasma cells (medullary cords) Afferent lymphatic vessels drain fluid from tissues & carry antigens & antigen-presenting Dcs from infected tissues to lymph nodes High endothelial venules (HEV) in paracortical area = entry portals for lymphocytes into lymph node DCs & T cells meet & interact in paracortical are. DCs short-lived & die there. Efferent lymphatics = exit routes for all lymphocytes from lymph node. Spleen: Red pulp: red blood cell destruction/disposal White pulp: immune compartment, lymphocytes around arterioles Periarteriolar lymphoid sheath: T cell zone in white pulp, site of DC-T cell interactions Follicles: B cell zone, germinal center surrounded by B cell corona & marginal zone Peyer’s patch: Subepithelial dome with DCs, T cells & B cells, no afferent lymphatics, antigen enters directly from gut across specialised epithelium made up of microfold (M) cells, lymphocytes enter across walls of HEVs & leave cia efferent lymphatic Macrophages: Long-lived, in all tissues, phagocytose & destroy bacteria or dead cells, in spleen macrophages of red pulp help degrad old red blood cells or immune complexes Granulocytes: Neutrophils: characteristic intracellular granules, short-lived, produced in bone marrow, rapidly recruited to sites of infection/inflammation, take up & kill pathogens Eosinophils & basophils: less abundant, granules contain many enzyme & toxic proteins, released when cells activated, defense against parasites & allergic response Mast cells: Begin development in bone marrow, migrate as immature precursors that mature in peripheral tissues (skin, intestines, airway mucosa), granules contain inflammatory mediators (histamine, proteases protect internal surfaces from pathogens like parasitic worms) Dendritic cells: Phagocytic when immature, activate T lymphocytes after maturation NK cells: Of innate immune system, lack antigen-specific receptors, express Fc receptors (if Ab binds to virally infected cell/tumour cell, NK destroys it), similarities with lymphoid lineages of adaptive immune system Chapter 2 – Innate Immunity: The first lines of defense Extracellular bacteria mostly cleared by phagocytes. Extracellular viruses prevented to adhere or enter host cells by antibodies. Intracellular viruses: infected cells attacked by NK cells/cytotoxic T cells. Intracellular bacteria/protozoa cleared by macrophages with help from T cells Mechanisms of tissue damage: directly through pathogens or indirectly through host defense Endotoxins: intrinsic components of microbes, trigger pathogen recognition receptors (PRRs) → most famous: Lipopolysaccharide (LPS) of outer cell membrane of Gram-negative bacteria → fever, rashes, pain, septic shock Exotoxins: secreted toxins released by microorganisms & act on host cell surfaces Epithelial surfaces = first barrier against infection Epidermis: Multiple layers of keratinocytes in different stages of differentiation from basal layer of stem cells, differentiated in stratum spinosum → β-defensins and cathelicidins in secretory organelles (lamellar bodies) into intercellular space to form waterproof lipid layer (stratum corneum) with antimicrobial activity Lung: Ciliated epithelium, beating moves mucus secreted by goblet cells outward, trapping & ejecting potential pathogens, Type II pneumocytes in lung alveoli produce & secrete antimicrobial defensins, mucins don’t have antimicrobial activity, only produce mucus Intestine: Goblet cells produce thick layer of mucus, Paneth cells in epithelial crypts produce antimicrobial proteins (α-defensins (cryptdins), antimicrobial lectin RegIIIα) Morbus Crohn: common inflammatory disease of intestines, inflammation caused by T cell response against antigens from commensal bacteria, associated with defective intestinal barrier function Lysozyme: Saliva, tears, also produced by phagocytes, digests cell walls of Gram- positive/negative bacteria (cleaves β-(1,4) linkage between N- acetylglucosamine and N-acetylmuramic acid, more effective in Gram- positive due to lack of LPS outer layer) Defensins: Short cationic peptides, amphipathic, disrupts cell membrane of microbes, produced in inactive form, activated by proteolytic cleavage RegIIIα: Family of bactericidal proteins produced by Paneth cells in intestine, C-type lectins, kills bacteria directly by forming hexameric pore in bacterial membrane, preferentially Gram-positive (peptidoglycan exposed, LPS of Gram-negative inhibits), proteolysis by trypsin activates in intestinal lumen Complement system System of soluble pattern recognition receptors & effector molecules detecting & destroying microorganisms, similarity to blood coagulation system (enzymatic cascade of protein activation, rapid amplification), activators & inhibitors of complement activation, several proteases (synthesised as zymogens = inactive pro-enzymes), products of cleavage reaction designated by adding lowercase letter as suffix (a=smaller, b=bigger) Activation by 3 different pathways (lectin: recognition of carbohydrae motifs, classical: recognition of Abs bound to pathogen, alternative) → C3 cleavage (exposes highly reactive thioester in C3b → covalent attachment to microbial surface or inactivation by hydrolysis) → 3 outcomes: - Production of C3a (& C5a = anaphylatoxins) → inflammation & leukocytes recruitment - Phagocytosis of C3b-tagged microorganisms by complemetn receptor expressing phagocyte - Lysis of microbes/cells on which complement activation (C5b deposition) took place Lectin pathway: Terminal mannose only in bacteria/yeast → recognised by lectins Triggered by binding of mannose-binding lectin (MBL, synthesised in liver, binds to mannose or fructose)/ficolins (synthesised in liver, lung & red blood cells, binds to oligosaccharide containing acetylated sugars) to microbial surfaces MBL monomer contains N-terminal collagen-like domain, α-helical neck region, C-terminal C-type lectin domain → 3 MBL monomers form trimer → 2-6 trimers form mature MBL molecule → MBL associates with MBL-associated serine proteases (MASPs) Binding induces MASP-1 to cleave & activate MASP-2 → cleaves C4 & C2 → C4b2a = C3 convertase Classical pathway: Homologous to lectin pathway, initiated by binding of C1 complex (C1q = pathogen/Ab sensor, 6 identical subunits with globular head & long collagen-like tails; 2 C1r & 2 C1s = serine proteases, binding to C1q → cleavage of C2 & C4 → C4b2a = C3 convertase) to surface- bound Abs Alternative pathway: Happens all the time, spontaneously activated → deposition of C3b on cell/ pathogen surface Amplification loop for C3b formation: alternative C3 convertase = C3bBb complex, with help of factor D Spontaneous hydrolysis of thioester bond in C3 in blood → C3(H2O) → short- lived fluid-phase C3 convertase by Factor B & D → further increase in chances of C3b deposition on cell surfaces & generation of alternative convertases by Factor B & D C3b on surface + C3 convertase → C5 convertase: cleaves C5 into C5a & C5b → C5b initiates assembly of terminal complement components → membrane-attack complex (MAC) generates pore in membranes (C7 & C8 conformational changes, expose hydrophobic domains to insert into membrane) Cell-surface complement receptors: CR1 → phagocytosis, C5a receptor→ G-protein-coupled receptors, enhance phagocytosis Opsonisation: decoration of pathogen surface with Abs/complement protein Anaphylatoxins (C5a & C3a) cause local inflammatory responses by acting on blood vessels: vascular permeability, upregulation of adhesion molecules; also activate mast cells → release inflammatory mediators (histamine & TNF-α) ➔ Large amounts cause anaphylactic shock Complement regulatory proteins: Help protect host cells from unwanted complement activation, in plasma & host-cell membrane, typically inhibit either activation C1q, C3 or C5 convertase activity or formation of MAC Example: Decay-accelerating factor (DAF) present on host cells & displaces C2a from C4b2a → block convertase activity; inhibition of activation of C1 complex; inhibition of C5 convertase activity (CR1 & H displace C3b, cofactors for cleavage of C3b by I); inhibiting assembly of MAC Pathogens produce inhibitors of complement activation: Staphylococcus aureus → protein A binds to Fc portion of Abs & inhibits complement activation & opsonisation → immune evasion & industrially exploited for affinity chromatography of therapeutic Abs Chapter 3 – The Induced Responses of Innate Immunity PRRs (pattern recognition receptors): - Free receptors in serum (MBL or ficolin) - Membrane-bound phagocytic receptors (mannose receptor) - Membrane-bound signalling receptors (TLRs) - Cytoplasmic signalling receptors (NOD receptors) Microbes recognised, ingested & killed by resident phagocytic cells (macrophages, dendritic cells), recruited phagocytes (granulocytes, inflammatory monocytes) → phagocytes & monocytes express high levels of PRRs → receptor interacting with microbial surface → internalised in phagosomes → fuse with lysosomes to form phagolysosome → pathogen destruction Bactericidal agents upon uptake of microorganisms: acidification, toxic oxygen-derived products (ROS), toxic nitrogen oxides (NO), antimicrobial peptides, enzymes (lysozyme), competitors Microbicidal respiratory burst: initiated by activation-induced assembly of phagocyte NADPH oxidase, bacterial fMLF & C5a receptors involved in generating reactive oxygen species (ROS) Neutrophils: Not tissue-resident cells, first recruited to site of inflammation from bloodstream, short-lived, high phagocytic capacity, dead & dying neutrophils = pus (Eiter), generate neutrophil extracellular traps (NETs, expelled chromatin) capturing microorganisms → more efficient phagocytosis Toll-like receptors: 10 expressed TLR genes in humans, each TLR recognises distinct PAMPs ➔ Sensors for microbes in extracellular spaces (cell surface receptors or intracellular in membrane of endosomes → phagocytosis) Structure: single-pass transmembrane proteins, extracellular region leucine-rich repeats (LRR), multiple LRR → ligand binding ➔ Formation of dimer or conformational changes in preformed TLR dimer TLR-3/7/8/9: viral RNA or bacterial DNA with unmethylated CpG motifs (9) → only released when pathogen taken up by cell & broken down → TLRs in intracellular compartments of phagocytes or B cells TLR-4: on cell surface, ligand = LPS of Gram-negative bacteria, already low levels in humans lead to septic shock due to overwhelming secretion of cytokines TLR signalling: Dimerisation of 2 TLR ectodomains brings cytoplasmic TIR domains together → cytoplasmic adaptor molecules interact with it & start signalling cascade: MyD88 (most important), links to NFκB activation Results in production of inflammatory cytokines, chemokines/chemokine receptors, antimicrobial peptides, Type I interferons (IFN-⍺ & IFN-β), costimulatory molecules Type I interferons: response to viral nucleic acids, TLRs 3,7,8,9 (nucleic-acid-sensing), signalling induces activation of transcription factors of interferon regulatory factor (IRF) family Cytosolic PRRs: NOD-like receptors (NLRs): In cytosol, contain nucleotide-binding oligomerisation domain (NOD), detect microbial products or cellular damage (fragments of cell-wall peptidoglycans), dimerise upon ligand binding, signalling → cytokine production via NFκB activation, mainly expressed in epithelial cells (barriers), macrophages, dendritic cells Morbus Crohn: inflammatory disease of intestine, develops T cells against antigens from commensal bacteria, mutations in NOD-2 (helps recognise invading bacteria early) → local transcytosis of microbiota into intestinal tissue → inflammatory response & T cell response → granulomatous inflammation NLRP proteins: Contain Pyrin domain NLRP3 activated by: ROS, reduced intracellular K+, high ATP, disruption of lysosomes, uric acid crystals → DAMPs = damage-associated molecular patterns Activation → formation of inflammasome, production of pro-inflammatory cytokines IL-1b & IL-18 & cell death through pyroptosis Sensors of intracellular infection & of cellular damage RIG-I-like receptors (RLRs): Detect viral RNA produced within infected cell, in many tissues, induce production of type I interferon & inflammatory cytokines (via NFκB & IRF3), discrimination for example by capping cGAS/STING: Viral, microbial, protozoan DNA in cytoplasm → dsDNA in cytosol → cGAS converts ATP & GTP to cGAMP → induces dimerization of STING (stimulator of interferon genes) → phosphorylation/activation of transcription factor IRF3 → production type I interferons Co-stimulatory molecules: activation of innate sensors in macrophages & DCs trigger expression, CD80 & CD86 most important & induced by TLR signalling, important for priming of naïve T cells by antigen presented on DCs, but it need high levels for proper T cell activation & induction of adaptive immunity Adjuvants in vaccines as PAMPs & DAMPs to induce MHC & costimulatory molecule expression in antigen-presenting DCs Tissue inflammation: Endothelial activation Inflammation to deliver effector molecules & recruitment of leukocytes to sites of infection, induce local blood clotting (physical barrier), promote repair of injured tissue Endothelial activation: increase in vascular diameter, expression adhesion molecules, increase vascular permeability, clotting microvessels; induced by inflammatory mediators (lipid mediators, chemokines & cytokines → TNF⍺, C5a) due to pathogen recognition by macrophages & neutrophils Leukocyte recruitment & extravasation: 0) Flowing (0.5-1 mm/s) 1) Rolling 2) Chemokine-mediated integrin activation & tight binding 3) Transmigration = diapedesis 4) Migration to site of infection, following chemotactic gradient Cell adhesion molecules: Selectins: induced on activated endothelium, initiated rolling → loose interaction Integrins: tight adhesion, expressed on leukocyte, 2 transmembrane protein chains (common β chain with different α chains), chemokine signalling induces switch to active state (high-affinity ligand binding), firmly bind to adhesion molecules of Immunoglobulin superfamily (ICAM-1 = Intercellular adhesion molecules, extracellular domain composed of immunoglobulin-like proteins) → upregulated in inflamed blood vessels → enhanced recruitment of leukocytes Chemokines: Small proteins inducing direct chemotaxis in leukocytes, response to PAMP & DAMPs, or constitutively particularly in lymphoid organs, signal via GPCRs → changes in cell adhesiveness & cytoskeleton → directed cell migration Function: - extravasation from blood vessels (integrin activation/firm adhesion) - chemotaxis: attract leukocytes - lymphocyte development, migration of developing cells, angiogenesis Tissue inflammation: production of cytokines Cytokine = small proteins involved in (immune) cell communication, usually released by cells due to activating stimulus, autocrine/paracrine/endocrine Families: interleukins (IL-1 inflammation & IL-2 T cell activation), interferons (IFN-α, IFN-β, IFN-γ), hematopoietins, tumor necrosis factor family (TNFα inflammation, lymphotoxin), chemokines Cytokine receptors: - Common γ chain receptors: for many IL, heterotrimeric receptors, mutations in γ chain → X- linked Severe Combined Immunodeficiency (no T, B and NK cells) - TNF receptors family: receptors & ligands homotrimers, signalling activates NF-κB, some co- stimulatory molecules, some induce apoptosis - Receptors activating JAK-STAT pathway: Tofacitinib inhibits JAK3/JAK1, treatment of rheumatoid/ psoriatic arthritis, block action of several inflammatory cytokines Macrophages produce inflammatory cytokines to recruit more cells, local & systemic effects contribute to innate & adaptive immunity TNF-α: Produced by activated macrophages, local inflammation → leukocyte recruitment & containment of infection Action on endothelial cells: upregulation of adhesion molecules, enhanced permeability (edema), blood coagulation High systemic concentrations: vasodilation & vascular permeability → loss of blood pressure → septic shock, systemic blood clotting in small vessels ➔ Balance important!!! TNF-α blockers: several antibodies (Humira), against psoriasis, psoriatic arthritis, rheumatoid arthritis, Morbus Crohn, best- selling biopharmaceuticals Corticosteroids: anti-inflammatory & immunosuppressant drugs, treat autoimmunity, allergy, upon organ transplantation, act on intracellular receptors & affect transcription of genes in leukocytes → pro-inflammatory functions of monocytes, macrophages & CD4+ T cells reduced, in blood vessels reduce adhesion molecules, chemokine expression & vascular permeability Acute-phase response: Infection → macrophages & DCs produce cytokines → neutrophil mobilisation, production of host-defense proteins in liver, fever (clinical symptoms, measured by doctor) TNF⍺, IL-1β and IL-6 = endogenous pyrogens LPS = exogenous pyrogen Acute-phase proteins (induced by TNF⍺, IL-1β and IL-6): C-reactive protein (binds phosphocholine of bacteria, opsonisation & activates complement cascade), mannose-binding lectin (MBL, complement activation) Interferon alpha (IFN-α): Type I interferon, synthesis by most cells when virally infected, mostly by plasmacytoid dendritic cells (pDC) Blocks spreading of viruses to uninfected cells:  - viral replication - MHC I expression - activates DCs, macrophages & NK cells As treatment of chronic Hepatitis-C/-B infection Natural killer cells: Recognise virally infected cells, activated by type-I interferons (IFN-α) & macrophage-derived cytokines Cytoplasmatic granules contain cytotoxic proteins similar to those of T cells, killing triggered by germline encoded PRRs recognising molecules on surface of infected/malignant cells Contain infections until adaptive immune response ready Express Fc receptors → binding of IgG Abs activates NK cells to release granules → Ab-dependent cellular cytotoxicity Different receptor families, all contain activating & inhibiting receptors, any given NK cell only a subset of receptor → not all NK cells in individual are identical All contain special signalling motifs in cytoplasmic domain Activating receptors: recognise cell-surface proteins induce in target cells by stress/damage → signalling activates killing, immunoreceptor tyrosine-based activation motif (ITAM), example: NKG2D with MIC-A & MIC-B as ligands (expressed when metabolic stress or infection), recognition = danger signal Inhibitory receptors: recognise surface molecules constitutively expressed at high levels (MHC class I molecules) → signalling inhibits killing (cell’s MHCI expression often reduced by virus!!!), immunoreceptor tyrosine-based inhibition motif (ITIM) ➔ balance between activating & inhibitory receptors determines whether NK kills target cell NK receptors ligand binding → Tyr residues in ITAM/ITIM phosphorylated → transmit activating/inhibitory signals into NK cells, balance of signals affect cytokine production & cytotoxicity Chapter 4 – Antigen Recognition by B-Cell Receptors & T-Cell Receptors Antibodies: Effector molecules of humoral immune response, on B cells as B cell receptor, constant & variable (binds antigen) immunoglobulin domains, 2 binding sites → avidity Structure: 2 heavy chains (50 kDa) + 2 light chains (25 kDa) linked by disulfide bonds, antigen binding site composed of variable heavy & variable light chain domain Isotypes (5): IgM, IgD, IgG, IgA, IgE → differ in distribution in body & effector functions, recognised by Fc receptor Folding of immunoglobulin domain: each domain consists of 2 anti-parallel β sheets (stabilised by internal disulfide bond) → β sandwich with loops = antigen- binding sites Fc fragment = fragment crystallisable, Fab fragment = fragment antigen-binding Hinge region → felxibility in Ab binding of multiple antigens Affinity = strength of interaction between binding site & antigen →KD Avidity = total strength of sum of interactions between Ab & antigen in all binding sites Antigen binding via hypervariable regions present in variable domains of heavy & light chain, other parts = framework Hypervariable regions = complementarity- determining regions (CDRs) → formed surface is complementary to antigen, in discrete loops in folded structure, 3 CDRs in heavy chain & 3 in light chain variable domain contact antigen → 6 in total ➔ electrostatic forces ➔ hydrogen bonds ➔ VdW forces ➔ Hydrophobic forces (excluding water molecules, pack together) ➔ Cation-pi interaction (non-covalent interaction between cation & electron cloud of nearby aromatic group) Abs can bind to conformational or linear epitopes (= part of antigen recognised by Ab) Antigen recognition by T cells : T cell receptor resembles membrane-bound Fab fragment, composed of TCR α & β chain, variable domains (Vα and Vβ) differ between different T cell clones, each T cell ca. 30’000 identical TCRs on surface Structure: Ig-like domain folding, 2 anti-parallel β sheets, framework variable domains can be superimposed with Ab variable domains, each TCR variable domain 3 CDR loops, some differences in constant domain folding & domain interactions TCR recognises antigen in complex of foreign peptide bound to MHC molecule ➔ MHC class I: all nucleated cells of body, present fragments of proteins expressed by itself (from cytosol), recognised  by cytotoxic CD8+ cells, important to detect viral infections of cells, highly on APCs & some other leukocytes ➔ MHC class II: antigen- presenting cells = APCs (DCs, macrophages, B cells, present fragments of proteins taken up into APC from outside, CD4+ T cells, also highly expressed on thymic epithelial cells (important for T cell development) ➔ Cross presentation: DCs present on MHC class I Structure MHC class I: 2 polypeptide chains, α chain (spans membrane, folded α1 & α2 form peptide binding cleft/groove → β sheet floor & 2 α helices as walls) & β2-micoglobulin Structure MHC class II: 2 polypeptide chains, α chain & β chain both span membrane, folded α1 & β1 form peptide binding cleft/groove → β sheet floor & 2 α helices as walls, α2 & β2 have immunoglobulin fold Peptides stably (non-covalently!) bound to MHC, bound peptide stabilises MHC, MHC I binds short peptides by both ends (8-10 aa, different genes & different versions which have different preferences for binding motifs but characteristic), MHC II binds peptides by several residues (13-17 aa → overhangs, anchor residues at various distances from end) ➔ Relevance of knowing peptide binding preferences of MHC: tumour immunology, organ transplantation, immunogenicity of therapeutic proteins TCR binds to peptide:MHC complex, binding involves TCR interactions with both peptide & MHC, mainly CDR loops of TCR, TCRs baseline affinity for MHC (peptide- independent) CD4 & CD8: CD8 = disulfide-linked heterodimer, MHC I CD4 = monomer (4 Ig-like domains), MHC II Binding contributes to overall effectiveness of T cell response → co-receptors (don’t bind at same place as peptide or TCR, help build right complex, bind framework = invariant sites, domains constituting peptide binding cleft vary) Increase sensitivity of T cell to antigen presented on MHC 100x Alternative TCR composed of γ & δ chain → γδ T cells in lymphoid organs & epithelial tissues, intermediate function between innate & adaptive immunity, limited receptor diversity → γδ T cell ligands not peptide:MHC complex, but in category of PAMPs/DAMPs, induced by cellular stress/infection Chapter 5 – The generation of Lymphocyte Antigen Receptors Primary immunoglobulin gene rearrangement In mature B cell variable domain of Ab encoded by single V-region exon (immunoglobulin fold, 9 β sheets), Ab binding-site formed by CDR1, CDR2, CRD3 or hypervariable regions HV1, HV2, HV3, CDRs separated by framework regions Different variable regions (VH & VL) not encoded in final version in germline, during development B & T cells rearrangement of gene locus encoding B or T cell receptor, variable region exons rearranged from V (CDR 1 & 2), D (only in VH) & J (CDR3 & last framework) segments → joint region in CDR3 → highly variable between different B cells Light chain C region in separate exon Heavy chain C region in multiple separate exons ➔ Joined to the V regions by splicing of mRNA after transcription Recombination process creating heavy chain V region in 2 stages: 1. DH (diversity) joined to JH 2. VH rearranges to DJH to complete VH region exon ➔ Variability of V regions of immunoglobulins by random selection of V (D) J segments Some segments accumulated mutations → non- functional protein, pseudogenes, rearrangements with pseudogenes = non-functional Genes organised into 3 clusters/genetic loci: heavy chain locus, κ or λ light chain loci → even more variability Heavy chain locus: Series of C regions arrayed one after the other, each corresponding to different isotype (IgM, IgD, IgG, IgE, IgA) → effector functions Rearrangement guided by conserved noncoding DNA sequences (heptamer & nonamer) = recombination signal sequences (RSSs), adjacent to points of recombination, usually 12 bp (1 turn in DNA double helix) with 23 bp (2 turns, sequences on same side of helix, allow interaction with proteins catalysing recombination) RSS joined → avoids joining of 2 V or V to J in heavy chain V-region recombination similar mechanism for H & L chain locus, looping out & deletion between 2 joined segment or retention of intervening DNA in chromosome Coding joint junction imprecise (nucleotides added/lost between joined segments → junctional diversity Enzymes: V(D)J recombinase, lymphoid-specific components = RAG-1 & RAG-2 encoded by recombination-activating genes RAG1 & RAG2 (only expressed when recombination in development, essential) Diversity of immunoglobulin repertoire: - Multiple versions of each V (D) J segment → different combinations - Junctional diversity as result of addition/subtraction of nucleotides (RAG proteins, Artemis, P-nucleotides, N- nucleotides, terminal deoxynucleotidyl transferase TdT) → could lead to frame-shift mutations → no real Ab → cells have to die - Different combinations of VH & VL forming antigen-binding site - When B cell activated, somatic hypermutation → point mutations into rearranged V region genes TCR gene rearrangement TCR α & β chains consist of V & C region, variable domains (Vα & Vβ) superimposed with Abs VH & VL, TCR gene segment arranged similarly as immunoglobulin gene segments & by same enzymes Gene segments organised into TCRα & TCRβ locus, rearrange during T cell development to complete V domain exons Similarities to immunoglobulin rearrangements: - RAG1/2, Artemis, TdT - CDR1 & CDR2 encoded in V gene segment, CDR3 created by V(D)J joining Difference: less diverse C region → only transmembrane polypeptides, only 1 Cα gene & 2 Cβ genes, but very closely homologous & functionally equivalent TCR binds peptide:MHC complex → less variable CDR1 & CDR2 loops of TCR mainly contact less variable MHC component of ligand, highly variable CDR3 contact unique peptide component γ:δ TCR: composed of γ & δ chains, organisation of loci similar to TCRα & TCRβ, δ encoding region within TCRα locus, rearrangement of Vα gene segment deletes intervening DNA encoding Vδ locus → T cell either develops into α:β or γ:δ T cell Structural variation in immunoglobulin constant regions Immunoglobulin C regions: Transmembrane or secreted Ab, heavy-chain locus encodes different C regions (CH) → isotype, CL only structural attachment for V region, no functional difference between λ & κ light chains TCR C regions: Support V regions & anchor receptor into membrane, no variation after assembly of complete receptor gene Isotypes differences: - Number of constant heavy chain domains - Presence of hinge region - Glycosylation site (IgGs only at one site on heavy chain) - Presence & location of disulfide bonds - Form of protection from infection → effector function Effector functions: neutralisation (IgA, IgG), opsonisation (IgG), complement activation (IgM, IgG), activation of mast cells (IgE), antibody- dependent cellular cytotoxicity (ADCC, IgG), transport across placenta (IgG)/into secreted fluids (gut, saliva, tears, IgA) IgM: IgM & IgD from same pre-mRNA transcript by alternative splicing, both expressed on surface of mature B cells, IgM first secreted immunoglobulin produce after activation, high molecular weight because secreted as pentamer, in blood but not tissues, low affinity compensated by high avidity, activator of complement IgD: Immediately 3’ to µ gene lies δ gene encoding C region of IgD, secreted in only small amounts by plasma cells, function still unclear IgG: Most abundant, 4 subclasses: IgG1, IgG2, IgG3, IgG4 → decreasing abundance, vary in effector function efficiency, all therapeutic Abs are IgGs, long half-life (2-4 weeks), across placenta IgA: in blood but also on mucosal surfaces, secreted into gut & respiratory tract, mother’s milk, monomer/dimer IgE: Extra CH instead of hinge-region, induce mast cell degranulation or activation of eosinophils & basophils, defense against multicellular parasites, allergic diseases B cells initially express transmembrane form of IgM → stimulation by antigen → some of progeny into plasma cell produce secreted Abs, differential splicing of mRNA regulates whether hydrophobic transmembrane region or hydrophilic secretory tail included Class switching: during B cell activation (shift in Ab production to IgA/IgE/IgG), requires irreversible changes to DNA IgM & IgA multimers by interacting with J chain (additional polypeptide chain) Dimeric IgA: monomers have disulfide bonds to J chains & to each other Pentameric IgM: monomers crosslinked with disulfide bond to each other & J chain Chapter 6 – Antigen Presentation to T Lmyphocytes Generation of α:β TCR ligand Cytosol & vesicular system = main intracellular compartments with antigens Cytosol: transported into ER & loaded onto newly synthesised MHC I Extracellular: taken up by endocytosis/phagocytosis into endosomes/phagosomes → fuse with lysosome, loaded onto MHC II → B cell activation & Ab production! Intravesicular: some pathogenic bacteria & protozoan parasites survive ingestion & replicate inside intracellular vesicles → MHC II Proteasome: degradation of damaged & unneeded proteins, forms channel (core & regulatory units), peptides generated from ubiquitinated proteins in cytosol Immunoproteasome: stimulation with interferons (viral infection) → catalytic subunits in constitutive proteasome exchanged → alters enzymatic specificity → more peptides with suitable anchoring residues for MHC presentation (MHC also induces by interferons) → more antigen presentation TAP = Transporter associated with Antigen Processing, TAP1 & TAP2 form membrane transporter in ER, ATP-catalysed transport of peptides cytosol → ER Mutations in TAP1/2: loading of MHC I in ER impaired, newly synthesised MHC I don’t reach cell surface → immunodeficiency = MHC class I deficiency MHC class I: only leaves ER when peptide bound → α chain only stable when correctly folded & in complex with β2-microglobulin & bound peptide (calnexin & chaperons help folding & loading) MHC class II: entered through endocytosis, phagocytosis, micropinocytosis, mainly expressed on APCs (B cells, macrophages, dendritic cells) MHC II molecules first delivered to ER → premature loading of endogenous peptides prevented by binding of invariant chain (CD74), cleaved into short peptide fragment = CLIP (placeholder until final peptide loading in acidified vesicles) HLA-DM in acidified vesicles with MHC II & facilitates dissociation of CLIP & exchange with other peptide Autophagy pathways can deliver cytosolic antigens for presentation by MHC class II Cross-presentation by specialised DCs: capture extracellular antigen & load antigen-derived peptides onto MHC I In case a virus that is best fought by cytotoxic CD8+ T cells doesn’t infect APC MHC & its function HLA (human leukocyte antigen): class I (HLA-A, -B, - C), class II (HLA-DR, -DP, - DQ)  Polymorphic: many different alleles (for many genes more than 1000 alleles, most quite frequent → high chances that 2 different alleles on both homologous chromosomes, particular combination of MHC alleles on 1 chromosomes = haplotype) Polygenic: 3 genes encoding MHC I & 3 MHC II ➔ Co-dominantly expressed (further increases likelihood that unrelated individuals differ in MHC) ➔ Cell can express up to 12 different MHC molecules Allelic variation predominantly in peptide-binding region Antigen-specific TCR recognises complex of antigenic peptide & one particular variant of self-MHC molecule = MHC restriction TCRs contact with MHC via CDR1 & CDR2 of V region genes (need to complement each other for binding, positive selection in T cell development in thymus) → polymorphism of MHC particularly affects aa residues in peptide binding cleft & TCR-contact region Generation of ligands for unconventional T cell subsets Unconventional subsets: γ:δ T cells or invariant NKT cells, intermediary between innate & adaptive, cell stress/damage/infection, recognise stress-induced proteins or lipids, ligands = non-classical MHC genes (MIC-A & MIC-B = MHC class I-like molecule, recognised by NKG2D; CD1 with hydrophobic channel for glycolipids → present microbial glycolipids & lipopeptide antigens) Chapter 7 – Lymphocyte Receptor Signalling General Principles of signal transduction & propagation Transmembrane receptors convert extracellular signal sinto intracellular events. → kinases (phosphorylate proteins, Ser/Thr), phosphatases (remove P-groups from Ser/Thr), phosphorylation (de-/activates proteins, generates site on proteins to bind), dephosphorylation (block/activates protein) Receptor tyrosine kinases: Receptors with non-covalently associated kinases: Signalling proteins interact with each other & lipid signalling molecules via modular protein domains Scaffold proteins: large proteins with many phosphorylation sites, bring many different signalling proteins together, membrane localisation Adaptor proteins: bring 2 different proteins together Small G proteins: downstream of tyrosine kinase-associated receptors, inactive → active by GEFs & binding of GTP, Ras (lymphocyte signalling, oncogene) Recruitment of signalling proteins to cell membrane: extracellular signals sensed by receptors at cell membrane (start of signalling) → membrane localisation of signal transducers via: scaffold & adaptor proteins, small G proteins with lipid modifications, protein domains recognising lipid motifs Turning off signalling: post-transcriptional control mechanisms → phosphorylation/dephosphorylation, ubiquitinylation (target degradation in proteasome/lysosome) Amplifying signal: activated kinase activates further kinase, actiated enzyme generate many products, second messenger Ca2+ Antigen receptor signalling & lymphocyte activation TCR complex = variable antigen-recognition proteins (TCR) + invariant signalling proteins (CD3) ITAM (Immunoreceptor Tyrosine-based Activation Motif): contains 2 Tyr, binding of cognate peptide/MHC to TCR induces phosphorylation of ITAM, TCR contains 10 ITAMs BCR complex = cell-surface immunoglobulin (recognises antigen but can’t generate signal) + 1 Igβ + 1 Igα, signalling started via phosphorylation of ITAM ITAM recruits signalling proteins with tandem SH2 domain (kinases, TCR: ZAP-70, BCR: Syk) Co-receptor CD4 or CD8: kinase Lck bound to cytosolic part of CD4, CD4 associates with TCR complex → Lck phosphorylates ITAMs Role of CD4 & CD8: stabilisation of TCR- MHC complex, phosphorylation of ITAMs Other receptors can pair with ITAM-containing chains & deliver activating signals ZAP-70 phosphorylates scaffold proteins LAT & SLP-76: ➔ Activation of transcription factors (T cell proliferation & differentiation) ➔ Increased metabolic activity (T cell proliferation & differentiation) ➔ Cytoskeletal rearrangements (T cell proliferation, Immune synapse formation) ➔ Enhanced integrin activation & adhesiveness (Immune synapse formation) Phospholipase C-γ: phosphorylation of scaffold proteins LAT & SLP-76, PLC-γ activated by phosphorylation → activation of TFs (NFAT, NFκB, AP1 → required for expression of interleukin-2) Cleaves inositol phospholipids to diacylglycerol (DAG, stays in membrane → activation AP1 & NFκB), inositol-triphosphate (IP3, opens Ca2+ channels → Ca2+ release from ER → binds to & activates calmodulin → complex binds & activates calcineurin (phosphatase) → dephosphorylates NFAT → translocates to nucleus → IL-2) NFAT essential for IL-2 transcription in activated T cells → blocking transcription or blocking autocrine IL-2 signalling inhibits T cell activation → therapeutic immunosuppression (Cyclosporin, Basiliximab) Cyclosporin: cyclic peptide of 11 aa, natural substance from fungus, immunosuppressant for treatment/prevention of transplant rejection, complex with immunophilin → inhibits activation of calcineurin → NFAT not activated & autocrine production of IL-2 reduced → suppressed T cell activation Akt: Ser/Thr kinase, promotion of cell survival (inhibiting cell death) & enhancement metabolic activity via mTOR (=mammalian target of rapamycin) TCR signalling → activated PI 3-kinase → PIP3 = membrane-docking & activation site for Akt (=protein kinase B) mTOR inhibitors: Rapamcyin, from bacteria, binds FK-binding protein → rapamycin-FKBP complex inhibits cell growth & proliferation by selectively blocking activation of kinase mTOR by Raptor Immune synapse: large zone of cell-cell contact stabilised by adhesion moleccules, peptide-MHC/TCR complexes & costimulatory molecules drawn into synapses, may last for >60 min → T cell proliferates & differentiates into effector cell Composition: cSMAC: inner ring, peptide-MHC/TCR complex & costimulatory molecules (many cytokines) pSMAC: outer ring, adhesion molecules for stabilisation  ➔ Enables effective signalling ➔ Targeted release of cytokines (for T cell differentiation) BCR signalling: Like TCR signalling but: Scr-family kinases associated with BCR & phosphorylates Tyr in ITAMs (Lck in TCR), Syk docks onto phosphorylated ITAMs & induces further phosphorylation & signal propagation (Zap-70 in TCR) ➔ Important in B cell development ➔ Less important for B cell activation Co-stimulatory & co-inhibitory receptors T-cell co-stimulatory protein CD28 transduces signal to enhance antigen receptor signalling pathway (phosphorylated → activates PI3K, PLCγ → activation of NFAT, NFκB, AP1), binds B7 IL-2: autocrine T cell survival factor CD40 = co-stimulatory receptor on B cells (TNF receptor superfamily), via TRAFs → activation of NFκB & Akt signalling CTLA-4: T cell-expressed inhibitory receptor for B7 molecules, induced on activated T cells, downregulates its activation, competes with CD28 for binding B7 (expressed by APCs) & has higher affinity & avidity → outcompetes CD28-B7 binding & inhibits costimulatory signalling via CD28, genetic knockouts/mutations in CTLA-4 connected to autoimmunity CTLA-4-blocking antibodies for anti-melanoma cancer therapy (induction of anti-tumour T cell responses, checkpoint inhibitors) ITIM: Immunoreceptor tyrosine-based inhibition motif, recruits inhibitory phosphatases (protein or lipid) Chapter 8 – Development of B & T lymphocytes Development of B lymphocytes In bone marrow rom hematopoietic stem cells (rearrangement of BCR, specialised microenvironment signals for development of hematopoietic cells, stromal cells for adhesion, growth & differentiation factors → multipotent progenitor MPP → common lymphoid progenitors CLP → NK cells, B & T cells) → migrate to peripheral lymphoid organs (activation, only when development completed) Factors produced by stromal cells: CXCL 12 (chemokine, retains precursors in bone marrow niche, sticks cell together), IL-7 & stem cell factor SCF (growth & survival factors for developing B cells), cell adhesion molecules (cellular support) Rearrangement of gene segments requires activity of RAG-1/2 enzymes (recombination activating genes), starts in early pro-B cell stage with heavy chain rearrangement → pre-B cell stage (critical stage!) Productively rearranged immunoglobulin gene immediately expressed as protein, rearragned heavy chain co-expressed with surrogate light chain Pre-BCR signalling inhibits further heavy-chain locus rearrangemet & enforces allelic exclusion: simultaneaous rearrangement of heavy chains on both alleles (maternal & paternal), unfinished heavy chain inhibited → prevents one B cell expressing 2 different BCRs Pre-B cell stage: much cell division (30-60x), after division light chain rearrangement (→ cells with same heavy chain can get different light chain, isotypic exlcusion: productive rearrangement of κ or λ block rearrangement of other type), nonproductive light chain rearrangements rescued by further rearranging (more success), if fail: apoptosis Central B cell tolerance: binding to self molecules in bone marrow can lead to inactivation/cell death of immature B cell (test for autoreactivity) Peripheral B cell tolerance: immature B cells if bind to many multivalent/soluble self antigens in first days after exiting periphery → apoptosis or become anergic (immunologically unresponsive), if bind low-affinity, non-corsslinking self-antigen → become ignorant; mature B cells passed transitional B cell stage (activated by antigen) Immature transitional B cells: short-lived, maturation & survival signals in B cell follicles in spleen (low-level tonic signalling via BCR, BAFF = cytokines produced by follicular DCs) → differentiation into mature follicular/marginal zone B cells, if not sufficient signals apoptosis Mature B cells upregulate surface IgD & become long-lived Benlysta (Belimumab): anti-BAFF antibody against systemic lupus erythematosus (auto-Abs against nuclear/cytoplasmic proteins & DNA → large immune complexes in blood, inflammation & damage) Development of T lymphocytes T cell progenitors from bone marrow to thymus, mature T cells to secondary lymph organs SLOs for activation Thymus: Cortex (immature thymocytes, cortical epithelial cells, macrophages), medulla (maturing thymocytes, medullary epithelial cells, DCs, phagocytic cells) Epithelial cells form network surrounding developing thymocytes, if fails to develop normally → DiGeorge’s syndrome (no mature T cells, fewer B cells & defective Ab production), nude mice (mutation in TF for terminal epithelial differentiation) Stages of α:β T cell development correlate with program of gene rearrangement, expression of cell- surface proteins, signalling proteins & TFs Goals of thymic education / T cell selection: - T cell able to bind to MHC to screen for presence of foreign antigen (MHC restriction) - Not autoreactive Immature thymocytes proliferate in cortex befor TCR rearrangement, ca. 98% apoptosis because no productive gene rearrangement/not MHC-restricted (no low-affinity binding to MHC)/bind autoantigens in thymus Stages: 1. Progenitor arrives in thymus (medulla) 2. Interactions with stroma → differentiation into double-negative thymocytes (CD4-CD8-) 3. Migrate to cortex, proliferate, differentiate into double-positive cells (CD4+CD8+) 4. Thymic selection → CD4+ (MHCII-restricted) or CD8+ (MHCI- restricted) single-positive thymocyte Thymocytes at different developmental stages in distinct parts of thymus Rearrangement of TCR β chain already in double-negative stage (DN2) → when rearranged double- positive stage (upregulate CD4 & CD8) → massive proliferation & rearrange α chain → when complete TCR expressed, positive & negative selection Positive & negative selection of T cells 10-30% of thymocytes have receptors with ability to interact with self peptide:self MHC complexes (present on thymic cortical epithelial cells) → receive survival signals = positive selection MHC that induces positive selection determines cell phenotype (CD8+/CD4+ expression) & differentiation into cytotoxic/helper T cell Negative selection: occurs in cortex & medulla, when T cells react strongly with ubiquitous self antigens → deleted AIRE (autoimmune regulator): transcriptional regulator in medullary stromal cells, facilitates transcription of tissue-specific proteins in thymus  T cells with newly arranged TCRs no sufficient binding strength to self-peptide:self MHC complexes on thymic epithelium → die by neglect, rest positively selected Excessively strong reactivity to self-peptide:self MHC complexes → apoptosis (negative selection) Alloreactivity = strong T cell response vs. non-self MHC, organ transplantation (T cells of recipient recognise non-self MHC on cells of grafted organ) → immunosuppressive drug (without, rejection) Chapter 9 – T-cell mediated immunity SLO function Sites of initiation of adaptive immunity, anatomical crossroads for interactions of antigens & lymphocytes - All similar cellular composition (T & B cells 90%, DCs, macrophages, stromal & endothelial cells - Similarly defined B & T cell zones - Distinct routes of antigen arrival (afferent lymphatics, blood, gut lumen) - Different cell entry/exit routes (blood/lymphatic vessels) Naïve T cells recirculate several times per day through SLOs searching for antigen → increase chances of encountering antigen (immunesurveillance) Entry into lymph nodes occurs through HEVs → migrate into T cell area to meet APCs (DCs), no cognate antigen: leaves lymph node via efferent lymphatics, cognate antigen: activated T cell proliferates & loses ability to exit (retained 3-5 days, increase cell numbers several 1000x → army of cells), later differentiated effector cells exit via efferent lymphatics High blood flow velocity → high sheer forces, extravasation: multistep adhesion cascade Rolling: L-selectin binds to glycosylated vascular adhesion molecules (low- affinity, fast on-/off-rate, many binding adhesion molecules expressed on surface of HEVs) Adhesion: Integrins (α & β subunit) activated to high affinity state by chemokines, bind in HEVs & inflamed blood vessels of peripheral tissues to ICAM-1, VCAM-1 Natalizumab blocks leukocyte extravasation by blocking α subunit on effector T cells → inhibits interactions with VCAM-1 & MadCAM-1, reduced leukocyte recruitment to inflamed CNS & intestine → MS & Crohn’s disease Same intravascular adhesion steps during extravasation at site of inflammation/infection, other chemokines & adhesion molecules → set of chemokines/adhesion molecules dictates which leukocytes extravasate Modulation of sphingosine 1-phosphate receptors (S1PRs) block emigration of T cells out of lymph node parenchyma even if activated → treat autoimmune disease MS (Fingolimod) Sphingosine 1-phosphate gradient mediates egress of lymphocytes from lymph nodes, S1P levels high in lymph (efferent lymphatics) Professional antigen-presenting cells Macrophages, DCs & B cells, constitutively express MHCII, can take up antigen DCs: conventional activate naïve T cells, plasmacytoid sense viral infections & produce interferons Cross-presentation: present phagocytosed antigen on MHC-I, induction of cytotoxic T cells Langerhans cells & dermal DCs take up antigen in skin & transport it via afferent lymphatic vessels to draining lymph node Upon encountering PAMPs → DCs 2 maturation steps: upregulation of CCR7 for migration towards CCL21-expressing lymphatics & lymph nodes & enhanced antigen processing; upregulation of MHC & costimulatory molecules to boost T cell activation in dLNs B cells: surface immunoglobulins to present antigen, B cell receptor- mediated uptake → enrich for presentation of antigen-derived peptides on MHCII, but less efficient in priming naïve T cells, since less co-stimulatory molecules than DCs Macrophages: results in activation of macrophages Priming of naïve T cells by pathogen-activated DCs Priming: first-time activation of naïve T cells, only possible upon encountering antigen on DC, in SLO Interaction T cells & DCs: immune synapse cSMAC: inner ring, peptide-MHC/TCR complexes & costimulatory molecules pSMAC: outer ring, adhesion molecules for stabilisation ➔ Effective signalling, targeted release of cytokines Activation 3 types of signals: peptide-MHC (specificity & activation), B7 molecules (CD80, CD86, co-stimulation required for survival), cytokines (differentiation into specific type of effector T cell) Autocrine T-cell survival signal: IL-2/IL- 2 receptor signalling, IL-2 & IL-2 α chain (CD25) expressed upon activation (TCR & co-stimulatory signalling CD28) Binding → accelerated cell cycling, modulating T-cell differentiation & enhancing proliferation ➔ Targeted by powerful immune-suppressive drugs (Cyclosporin, Basiliximab) CTLA-4: inhibitory receptor for B7, competes with CD28 & higher avidity → wins, blocks T cell activation → CTLA-4-blocking antibodies for cancer therapy (anti-tumor T cell responses) Properties of effector T cells & their cytokines CD8+: cytotoxic T lymphocytes (CTLs) CD4+: TH1, TH2, TH17, TFH, Tregs Activation of T cells changes expression of cell surface molecules → altered trafficking behaviour & tissues tropism → less homing to lymph nodes (L-selectins), more to peripheral tissues TH1: fate specified by IFN-γ, IL-12, signal through IFN-γ, lead to macrophages kill intracellular microbes TH2: fate specified by IL-4, signal through IL-4/5/13, eosinophil, mast cell, basophil against Helminth parasites TH17: fate specified by TGF-β, IL-6/23, signal through IL-17/22, neutrophils against extracellular bacteria & fungi TFH: fate specified by IL-6, signal through IL-21, isotype switching & affinity maturation of B cells Tregs: fate specified by TGF-β, IL-2, inhibit DCs → no T-cell activation Differentiation-inducing cytokines activate different STAT family TFs → induction of lineage-specific TFs Cross-regulation between CD4 T-cells through cytokines: Cytokines present at early stage of infection determine which TH cell subset induced → specialised T cells help fight different types of pathogens T-cell mediated cytotoxicity CD8+ cytotoxic T cells recognise cells infected with intracellular pathogens, respond to target without co-stimulation Activation: simultaneous interaction of CD4+, CD8+ T cell & APC, antigen- specific CD4+ T cell helps by upregulating co-stimulatory molecules in APC & synthesising IL-2 for activation of CD8+ T cell T cell cytotoxicity need immune synapse with target cells → specific recognition of antigen on target cell induces T cell polarisation → effector molecules released towards target cells ➔ Apoptosis: programmed cell death, destroy from within, chromatin condensation & DNA fragmentation, cell shrinking, blebbing ➔ Necrosis: induced by hypoxia, physical damage, Ab/complement activation, bursting cell wal, release lysosomal enzymes → auto-digestion Lytic granules of cytotoxic T cells contain protein that trigger apoptosis (perforin, granzymes), same mechanism/molecules in NK cells Granzyme: activates Caspase-3, induces DNAse activity → DNA fragmentation → apoptosis Only kills target cells bearing specific antigen receptors but spare neighbouring cells, serial killers Chapter 10 – The Humoral Immune Response B cell activation by antigen & helper T cells BCR internalises antigen & induces signalling, second signal required: Thymus-dependent antigens: protein antigens alone, help from T helper cells (CD40-CD40L) → follicular helper cells Thymus-independent antigens: mostly microbial constituents, together with TLR ligand, arrayed antigens → cluster BCRs, only IgM responses → rapid first protection T follicular helper cells: cell-surface bound (CD40L with CD40 on B cell handshake, costimulation) & secreted (IL-21 & other cytokines induce proliferation, differentiation & class switching) signals activate B cells & control differentiation T cells & B cells recognise antigen in same molecular complex, but antigen recognised by TCR must not be the same that recognised by BCR, T cell antigen also taken up by B cell & presented on its MHC → T cell helps activate B cell & antibody production → linked recognition (2 cells have to check it, prevent autoimmune responses) Activated B cells → Ab secreting plasmablasts (intermediary stage) & plasma cells (terminally differentiated B cells, class switching, can’t proliferate or respond to antigen) Activation: B cell encounters antigen → moves to border of T cell area, interacts with antigen-specific T cell → proliferation & differentiation into plasmablast in primary focus → some back into follicle to form germinal center (sites of proliferation & differentiation, somatic hypermutation → antibody affinity maturation & class switching) → plasma cells remain in lymph node or migrate to bone marrow V(D)J joining → specificity for antigen (bone marrow) Somatic hypermutation → improve affinity for antigen (germinal centres) Class switching → isotype (germinal centres) Germinal centres: intense cell proliferation & cell death, specialised microenvironment for B cell proliferation, somatic hypermutation, affinity maturation, dark zone (proliferation & differentiation, somatic hypermutation) & light zone (testing, capture antigen on FDCs) → B cells cycle (acquire mutations), migration controlled by differential expression of chemokines & up/downregulation of corresponding chemokine receptor on B cells Follicular dendritic cells (FDCs): specialised stromal cells in B cell follicles, no hematopoietic origin, express Fc & complement receptors → bind antigen on surface, early formed or from previous immune response Abs bind these antigens to FDCs, for long time periods AID (activation-induced cytidine deaminase): initiates DNA lesions, uridine can trigger mismatch repair or base-excision repair → point mutations Expressed in germinal centre B cells, deamidates cytidine to uridine, only active on single- stranded DNA in actively transcribed loci (Ig locus), uridine foreign → mismatch repair & excision programs, here DNA polymerase is error-prone → mutations Somatic hypermutation introduces mutations into rearranged immunoglobulin variable regions to improve antigen binding (in CDR region) → closely related B cell clones that subtly vary in affinity, most mutations negative impact on BCR to bind antigen → apoptosis (specialised macrophages to remove them), if mutation improves affinity for antigen → survival advantage → expanded/selected Selection of high-affinity mutants in light zone = germinal centre reaction: high affinity B cells have advantage in presenting antigen to TFH & receiving survival signals → selected & return to dark zone for next round of mutations Class switching = isotype switching: constant region of Ab heavy chain is changed, first isotypes IgM & IgD (expression determined by RNA splicing) → all other isotypes require irreversible DNA recombination, in activated B cells & needs T cell help, AID, UNG & APE1 induce nicks in switch regions upstream of Cµ & target constant region → intervening DNA region deleted TFH interacts with B cells to determine choice of isotype → CD40-CD40L & IL-21 + other cytokines which determine → germinal centre B cells differentiate into Ab-secreting plasma cells & memory B cells (long lived, hardly divide, some surface Abs, rapidly activated during antigen re-challenge) ➔ Determines type of immune response, because different effector functions Distributions & functions of immunoglobulin classes Isotype determines type of immune response & distribution of Ab IgA: most abundant in lumen of gut, neutralisation, transported in dimeric form across mucosal epithelia → polymeric immunoglobulin receptor binds Fc regions of IgA & transports it FcRN Receptor (neonatal Fc receptor): transports IgG across placenta & recycles it in blood → long half-life, expressed in endothelial cells, monocytes, podocytes, proximal tubular epithelial cells Antibody distribution: IgM: intravascular IgA: mucosal surfaces (gastointestinal tract, nasal cavity, saliva, tear fluids) IgE: epithelial surfaces (GI tract, skin, lung, nasal cavity) IgG: all tissues within body (except uninflamed CNS) Neutralisation: Of toxins: prevent cellular damage (diphtheria, pertussis, tetanus vaccination) Of viruses: prevent infection (influenza, polio vaccination, SARS-CoV-2) Of adhesion: prevent bacterial colonisation Opsonisation: Coating pathogens with Abs & complement C3b → initiates phagocytosis, aggregation/clustering induces cellular activation (only if several Abs bind, allows cross-linking of Fc receptors) Complement activation: IgM very efficient at activating complement, can lead to formation of membrane-attack complex → cell lysis Antibody-dependent cell cytotoxicity (ADCC): Mediated by NK cells expressing FcγRIII, mechanism of action of several therapeutic antibodies Rituxan: antibody directed against B cell surface protein → destruction of B cells by NK cells, treatment of malignant B-cell tumours IgE-Response: Expel parasites, not kill, IgE-crosslinking on surface of mast cells → rapid release of granules containing histamine/other inflammatory mediators & secretion lipid mediators → allergy & protection against helminths Chapter 11 – Integrated Dynamics of Innate & Adaptive Immunity Integration of innate & adaptive immunity in response to specific types of pathogens Innate & adaptive immunity needed to overcome infection Different types of infections require different types of T helper cells Innate lymphoid cells (ILCs): From common lymphoid precursors but lack specific antigen recognition receptors, subgroups defined on basis of cytokine production (NK cells also subset), live in tissues, always responsible for one type of T helper cell Innate sensor cells produce cytokines that activate ILCs: amplify & coordinate innate response (clear many infection only non-adaptively, buy time for adaptive), influence development of distinct type of helper cells → produce same cytokines as corresponding TH subtype but earlier during immune response Group 1: activate macrophages to kill intracellular pathogens, TH1 differentiation & production of opsonising IgG Group 2: recruit eosinophils, mast cells, basophils, TH2 differentiation & production of IgE Group 3: production & recruitment of neutrophils, TH17 differentiation & production opsonising IgGs ➔ ILCs amplify/translate cytokine signal from innate sensor cells! Functions of different effector cell types TH1: fight intracellular bacteria/protozoa TH2: fight parasites (Helminths) TH17: fight extracellular bacteria & fungi TFH: support B cells Tregs: suppress overshooting/autoimmune response Cytotoxic CD8+ T cells (CTLs): kill virally infected cells & tumour cells TH1: many intracellular pathogens survive in macrophages (prevent fusion of phagosome & lysosome), pathogen-specific TH1 recognises peptide on MHCII of infected macrophage → secretes IFNγ & expresses CD40L → both required for full macrophage activation → phagosome/lysosome fusion & pathogen destruction TH1-activated macrophages: enhanced antimicrobial effectiveness of macrophages & further amplification of immune response, ROS production, secretion TNFα & IL-12, upregulation MHC & costimulatory molecules Chronic TH1-activation: granulomas (contain intracellular pathogens, can’t be cleared, core of infected macrophages & rim of T cells, wall-off pathogens), resistant intracellular bacteria & protozoa to microbicidal effects of activated macrophages → chronic low-level infection, if TH1 response defective → systemic spread of disease (fatal) TH2: coordinate type 2 response to expel (diarrhoea, vomiting etc) intestinal helminths (1mm-1m, too big to be killed/engulfed by single leukocyte) & repair tissue injury (limit damage) → high IgE levels by B cells (mast cell degranulation, release of cytotoxic granules by eosinophils), recruitment of eosinophils, mast cells, macrophages, TH2 cells, epithelial repair & mucus production Allergy = adaptive immune response against innocuous foreign substance, activation TH2 & IgE (cross- link on mast cells → granules containing histamine & inflammatory mediators & secretion of lipid mediators) Anti-allergic drugs: anti-histamines (block histamine receptors), mast cell stabilisers (block degranulation) TH17: coordinate type 3 responses & enhance clearance of extracellular bacteria & fungi, activated TH17-cells secrete IL-17 (→ neutrophils), IL-22 (→ antimicrobial peptides), induce production of pathogen-specific Abs (opsonisation ➔ Effector CD4+ T cells demonstrate plasticity & cooperativity enabling adaption ➔ Immune responses not unimodal, effector T cells transition into different cytokine production phenotypes (mostly in type 3, example: salmonella survive extracellularly & in macrophages) Lymphocyte trafficking Naïve T cells: adhesion molecules & chemokine receptors for homing SLOs Effector T cells: express trafficking molecules for extravasation at site of inflammation/infection → set of adhesion molecules & chemokine receptors depend on place they home, vasculature of this place expresses matching set (postal code) Immunologic memory After infection cleared, most effector T cells apoptosis, some plasma B cells remain & produce Abs, very few specialised memory T & B cells remain (can persist in absence of antigen) → more rapid responses & higher affinity (can re-enter germinal centres & undergo additional somatic hypermutation & affinity maturation) & higher amount of Abs Memory T cells divided into different categories based on trafficking characteristics; arise from effector T cells maintaining sensitivity to IL-7/IL-15, IL-7 = pan memory T cell survival factor (expressed by stromal cells in lymphoid organs, required for survival of naïve T cells & memory cells), IL-15 = CD8+ memory T cell survival factor