C8116 Immunochemical techniques Immune system, part I Spring semester 2024 Hans Gorris Department of Biochemistry February 20th, 2024 2 Research and contact Assoc. Prof. Hans H. Gorris Department of Biochemistry C05, office 315 Phone: 3816 E-mail: gorris@mail.muni.cz Our research focus: 1) Analytical biochemistry: - luminescent nanoparticles (UCNP) - single-molecule / digital immunoassays 2) Single molecule studies of enzymes: - single enzyme molecules in microchambers (50 fL) - structure-function relationship of enzymes => More information provided during the lecture… 3 Awarding reserach Novozymes: the world‘s market leader for the production of industrial enzymes: Enzyme Assay Scientist Award 2016 https://www.novozymes.com/en/news/news-archive/2016/09/award 4 Overview of the lecture Date Topic 1 Feb. 20th Immune system I 2 Feb. 27th Immune system II no lecture March 5th - 3 March 12th Antibodies as immunological tools 4 March 19th Immunoassays I 5 March 26th Immunoassays II no lecture April 2nd no lecture April 9th - 6 April 16th Immunoaffinity techniques 7 April 23rd Guest lecture: Prof. Tero Soukka 8 April 30th Advanced microscopy I 9 May 5th Advanced microscopy II 10 May 14th Advanced microscopy III 11 May 21st Advanced microscopy IV 12 find 1 additional date Electron microscopy During exam period Individual oral exams (30 min) 5 Guest lecture on April 23rd Prof. Tero Soukka University of Turku, Finland Department of Life Technologies/Biotechnology 1 pm: Evolution of lanthanide-based labels for immunoassays 2 pm: Research talk open for all 6 The idea behind the lecture Immunology The “tools“: antibodies Immunoassay 7 The immune system (2 days) 1) Innate / adaptive immune system 2) Lymphoid organs 3) B cells 4) Progress of immune response 5) Structure of IgG / immunoglobulin superfamily 6) Binding sites of antibodies 7) Generation of antibody diversity / affinity maturation 8) Antibody affinity 9) Clonal selection theory / immunological tolerance 10) Antibody classes IgG, IgM, IgA, IgE 11) Complement system 12) B cells vs. T cells 13) T-cell receptor 14) MHC class I and II 15) Antigen presentation 16) Cytotoxic / helper T cells 8 Recommended reading Basic text book Molecular Biology of the Cell (6th edition) Alberts, Johnson, Lewis, Morgan, Raff, Roberts & Walter Garland Science, London 2014 Chapter on Immunity New (7th) edition Slides of the lecture are available online (Learning Materials) In depth reading Immunobiology (7th edition) Murphy & Weaver Garland Science, London 2017 In online folder 9 Overview on our body‘s defenses against an infection 10 Challenge: Great variability of infectious diseases Cholera: bacterium, in the intestine Toxoplasmosis: single-celled, eukaryotic parasite, intracellular Nematodes: multicellular, eukaryotic parasite, in intestine, blood und lung Coronavirus, intracellular Diameter: ca. 100 nm 11 Infectious diseases Source: The World Health Report 2000, WHO Acute respiratory infections 4.0 million HIV/AIDS 2.7 million Diarrhoeal diseases 2.2 million Tuberculosis 1.7 million Malaria 1.1 million Measles 0.9 million Others 1.5 million Deaths per year red colors: Pathogens that enter our body over mucosal surfaces 12 Surface areas of human body 400 m2 Mucosa 2 m2 Skin 13 Two lines of defence Foreign and own structures, microorganisms, viruses, tumorsAntigen Innate immunity Adaptive immunity Neutrophil granulocytes, macrophages, NK cells, diverse humoral factors Antigen-presenting cells, T and B lymphocytes, plasma cells, cytokines T B + + - + - Immediately* up to 14 days antibodies + *Race against time: Bacteria can divide every 20 min xt=x0*e(µ*t) (µ=ln2/time for division (hrs)) => unrestricted growth: 1*e2.08*10= 1 billion bacteria after 10 hrs! 14 Innate / adaptive immunity Innate immunity Physical barriers Cellular defence (Bio-)chemical barriers immediate activity skin (2 m2) / mucosae (400 m2) Macrophages pH (gastric acid) Killer cells Lipids Enzymes (e.g. lysozym) Complement system Adaptive immunity Cellular immunity Interleucins Humoral immunity delayed activity (up to 14 days) T cells B cells => antibodies Act over short range (cell-cell contact) Act over long range (circulating antibodies) 15 Adaptive immunity: Human lymphoid organs Primary lymphatic organs (yellow): Bone marrow: B-cells Thymus: T-cells Secondary lymphatic organs (blue): lymph nodes spleen and others 1012 lymphocytes (ca. 1 kg) 16 Development of B und T cells => Bone marrow donation to reconstitute the immune system 17 Lymph node 18 Circulation of lymphocytes 19 Overview of an inflammatory response 1) A bacterium encounters a first line of defense (innate immune response) 2) Breakdown of bacterium and release of antigens 3) Dendritic cells take up antigen and activate T cells 4) T cells proliferate and activate B cells 5) B cells differentiate into plasma cells 6) Plasma cells produce antibodies 7) Antibodies neutralize bacterium 20 Two classes of adaptive immune responses => mediated by lymphocytes short-range action far-range action An adaptive immune response is unique for vertebrates / delayed activity (up to 14 days) both must be eliminated 21 Activation of lymphocytes => production of antibodies (500 antibody molecules/sec!) cytotoxic T cells helper T cells regulatory T cells 22 Progress of immune response 23 Immunological memory Vaccination! 24 B cells and antibodies 25 Structure of IgG Þ 2 light chains (25 kDa, light green) Þ 2 heavy chains (50 kDa, dark green) total mass: 150 kDa Two identical antigen binding sites SDS polyacrylamide gel stained with Coomassie 4 different IgG antibody clones against the same antigen 26 Structure of IgG Immunoglobulin domainsFragmentation of antibody F: fragment ab: antigen binding c: crystallizable (constant) 27 Immunoglobulin (Ig) superfamily Shown: important membrane-bound molecules of the immune system more than 750 members in total (also cell-cell interactions); many cell surface proteins 28 Membrane-bound BCR and secreted antibodies B cell receptor (BCR) “monoclonal antibodies“ a single clone of B cells 29 The hinge region => higher flexibility 30 monoclonal antibodies polyclonal antibodies Interactions of antibody and antigen 31 Multiple antigenic determinants: epitope 32 Antigen-binding sites of antibodies Different antigenic structures 33 Non-covalent binding forces [AgAb] 34 anti-parallel β sheets form a β barrel 4 strand + 3 strand 4 strand + 5 strand C‘ and C‘‘ are not present in the C region Detailed structure of antibody 35 Hypervariable regions of binding sites 36 Hypervariable regions of binding sites 37 Generation of antibody diversity: light chain κ light chain 40 V gene segments 5 J gene segments => 200 different combinations This happens before (and indpendent of) any contact with antigen! 38 heavy chain 40 V gene segments 25 D gene segments 6 J gene segments => 6000 combinations Generation of antibody diversity: heavy chain 39 Gene segment joining 40 200 350 6000 => about 2.000.000 combinations Generation of antibody diversity 41 Affinity maturation of antibodies Somatic hypermutation by activity-induced deaminase (AID) => 1 mutation per V region per cell cycle Cytosin: forms 3 hydrogen bonds Uracil: forms 2 hydrogen bonds 42 Main mechanisms of antibody diversity Þ There is an even larger repertoire of combinations than the 1212 existing B cells. After contact with antigen 43 Antibody affinity limits during immune responses Binding rate kon: 105-106 M-1s-1 => controlled by diffusion Release rate koff: 10-3-10-4 s-1 => controlled by time for signal transduction/endocytosis after antigen binding to cell surface receptors Maximum affinity* of antibodies: Ka = kon/koff = 1010 M-1 => Higher affinity antibodies may arise but would have no selective advantage (affinity ceiling) *for comparison: biotin-strepatividin: Ka = 1014 M-1 44 Clonal selection theory There’s only a single antibody specificity per B cell => mass production 45 Immunological tolerance But this system is not perfect: autoimmune diseases e.g.: Eppstein-Barr virus is suspected to induce multiple sclerosis 46 Theoretical considerations of antigen recognition Innate immune response: => Elimination of everything that is recognized as foreign (e.g. PAMPs) Problem: through natural evolution, a pathogen can adapt to hide or change its distinct antigenic signatures (pathogens have a big evolutionary advantage because they have a much shorter lifecycle (bacteria > 20 min) and larger populations than animals (> 1 year) in principle they can adapt 30,000x faster!) Time to acquire 2% difference in genome sequences Humans: 8 million years Poliovirus: 5 days Adaptive immune response: => Elimination of anything that is not recognized as own Solution: each individual person starts its mini-evolution within its leucocytes (instead of a whole life cycle, a pathogen-specific immune response is ready in less than 2 weeks) 47 Antibody classes Heavy chain defines the class of antibody Same light chain: κ or λ => B cells can switch between the production of antibody classes primary secondary classes of antibody 48 Class switch mediated by DNA rearrangement Class switch DNA recombination (not splicing!) => irreversible depends on switch sequences (consisting of tandem repeats) and the enyzmes activiation indcued deaminase (AID) + uracil-DNA glycosylase (UDG) 49 IgM: First antibody class on cell surface circulating in blood 50 IgG: Main class in blood 51 Opsonization TEM image 52 IgA: Defence of mucosal surfaces Þ Similar mechanism of IgG transcytosis across the placenta to protect the fetus Transcytosis 53 IgE: Protection against large parasites 54 Histamin Protection against parasites Allergies Release of histamin by mast cells 55 Classic experiment => Behring/Kitasato (ca. 1890) 56 Complement system => A protease cascade: amplification steps Covalent attachment / deposition on target cell 57 Lectin pathway 58 Complement system: pore formation/lysis