C8116 Immunochemical techniques Immune system, part I and part II Spring semester 2025 Hans Gorris Department of Biochemistry February 18th and 25th, 2025 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 180px-ELISA The idea behind the lecture Immunology The “tools“: antibodies Immunoassay 4 Topics of the lecture Part A: The immune system Part B: Antibodies as immunological tools Part C: Immunoassays Part D: Immunoaffinity and other protein-protein affinity techniques Part E: Advanced fluorescence microscopy for (life) cell imaging 5 The immune system (2 days) 1) General introduction to the immune system 2) Innate / adaptive immune system 3) Lymphoid organs 4) B cells 5) Progress of immune response 6) Structure of IgG / immunoglobulin superfamily 7) Binding sites of antibodies 8) Generation of antibody diversity / affinity maturation 9) Antibody affinity 10) Clonal selection theory / immunological tolerance 11) Antibody classes IgG, IgM, IgA, IgE 12) Complement system 13) B cells vs. T cells 14) T-cell receptor 15) MHC class I and II 16) Antigen presentation 17) Cytotoxic / helper T cells 6 Recommended reading Basic text book Molecular Biology of the Cell (7th edition) Alberts, Heald, Johnson, Morgan, Raff, Roberts & Walter W.W. Norton & Company, New York 2022 Chapter 24: The innate and adaptive immune system (page 1353-1404) https://archive.org/details/alberts-molecular-biology-of- the-cell-7th/page/1353/mode/2up Slides of the lecture are available online (Learning Materials) In depth reading Janeway Immunobiology (9th edition) Murphy & Weaver Garland Science, London 2017 https://inmunologos.wordpress.com/wp- content/uploads/2020/08/janeways-immunobiology-9th- ed_booksmedicos.org_.pdf 7 Overview on our body‘s defenses against an infection 8 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 9 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 via mucosal surfaces 10 Surface areas of human body 400 m2 Mucosa 2 m2 Skin 11 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! 12 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. lysozyme) 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) 13 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) 14 Development of B und T cells => Bone marrow donation to reconstitute the immune system (brzlík) 15 Lymph node 16 Circulation of lymphocytes 17 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 18 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 19 Activation of lymphocytes => production of antibodies (500 antibody molecules/sec!) cytotoxic T cells helper T cells regulatory T cells 20 Progress of immune response 21 Immunological memory Vaccination! 22 B cells and antibodies 23 Structure of IgG  2 light chains (25 kDa, light green)  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 24 Structure of IgG Immunoglobulin domainsFragmentation of antibody F: fragment ab: antigen binding c: crystallizable (constant) 25 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 26 Membrane-bound BCR and secreted antibodies B cell receptor (BCR) “monoclonal antibodies“ a single clone of B cells 27 The hinge region => higher flexibility 28 monoclonal antibodies polyclonal antibodies Interactions of antibody and antigen 29 Multiple antigenic determinants: epitope 30 Antigen-binding sites of antibodies Different antigenic structures 31 Non-covalent binding forces [AgAb] 32 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 domain Detailed structure of antibody 33 Hypervariable regions of binding sites 34 Hypervariable regions of binding sites 35 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! 36 heavy chain 40 V gene segments 25 D gene segments 6 J gene segments => 6000 combinations Generation of antibody diversity: heavy chain 37 Gene segment joining 38 200 350 6000 => about 2.000.000 combinations Generation of antibody diversity 39 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 40 Main mechanisms of antibody diversity  There is an even larger repertoire of combinations than the 1212 existing B cells. After contact with antigen 41 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 42 Clonal selection theory There’s only a single antibody specificity per B cell => mass production 43 Immunological tolerance But this system is not perfect: autoimmune diseases e.g.: Eppstein-Barr virus is suspected to induce multiple sclerosis 44 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) 45 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 46 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) 47 IgM: First antibody class on cell surface circulating in blood 48 IgG: Main class in blood 49 Opsonization TEM image 50 IgA: Defence of mucosal surfaces  Similar mechanism of IgG transcytosis across the placenta to protect the fetus Transcytosis 51 IgE: Protection against large parasites 52 Histamin Protection against parasites Allergies Release of histamin by mast cells 53 Classic experiment => Behring/Kitasato (ca. 1890) 54 Complement system => A protease cascade: amplification steps Covalent attachment / deposition on target cell 55 Lectin pathway 56 Complement system: pore formation/lysis 57 T cells and T cell receptor (TCR) 58 B and T cell maturation follow a similar course 59 We take a larger picture: Antigen presentation 60 Larger picture: initiation of immune response 61 Better double check! B cell epitope BCR T cell eptiope TCR recognition recognition  Minimizing the risk of wrong classification (friend/foe) to prevent e.g. autoimmune diseases, allergies 62 T cell receptor (TCR) 63 Generation of TCR diversity α-chain 70-80 V gene segments 61 J gene segments => 5000 combinations β-chain 52 V gene segments (2 D gene segments) 13 J gene segments => 700 combinations 64 Generation of TCR diversity Unlike BCR no somatic hypermutation => οnly lower affinity (Ka = 105-107 M-1) => Same enzymes are used for the gene rearrangement as in B cells => about 3.500.000 combinations 65 T cell activation A TCR recognizes the antigen only in context of an MHC 66 Major histocompatibility complex (MHC) => More information in online folder / doi: 10.1111/tan.14626 67 Major histocompatibility complex (MHC) Polymorphic region Class I MHC protein => on (almost) all cells Class II MHC protein => Only on professional antigen-presenting cells (e.g. dendritic cells) 68 Peptide bound to MHC 69 Peptide bound in the groove of MHC Peptides bound to MHC-I: 8-10 amino acids long Peptides bound to MHC-II: 10-12 amino acids long 70 Allelic variation in MHC genes Red: peptide binding regions 71 Human MHC genes There is a large variability of MHC molecules in a population, but in each individual: MHC-I MHC-II 2x HLA-A 2x HLA-DP 2x HLA-B 2x HLA-DQ 2x HLA-C 2x HLA-DR => 6 => 6-8 72 Structural comparison: antibody, MHC and TCR MHC I MHC II 73 Large diversity in the recognition of antigens BCR and antibodies: gene rearrangement + somatic hypermutation => Each individual can recognize any hapten/epitop (linear and conformational epitopes) TCR: gene rearrangement => Each individual can recognize any linear peptide in context with MHC molecule MHC: no gene rearrangement but 3 genes and several thousand alleles in a population => Can bind a large variety of peptides (but not all) => a whole population is well protected but there is an individual risk of missing some pathogenic peptides => populations with a large gene pool are more resistant to an epidemic 74 Interaction of TCR with a peptide on MHC cIass I => Only linear peptide epitopes 75 Friend / foe recognition by T cells 76 T cell recognition of antigens is MHC restricted 77 T cell recognizes viral antigen and host target cell 78 Co-receptors on T cells Serves as a receptor for HI virus binding 797979 => depletion of TH cells: AIDS (Aquired Immunodeficiency Syndrome) Excursion: HI virus infecting T cell 80 Classification of T cells T helper cells (TH) (CD4+ cells) => recgonize peptides on MHC II Cytotoxic T cells (TC) (CD8+ cells) => recognize peptides on MHC I TH1 infections by microbes TH2 infections by parasites Suppressor T cells regulatory function TH0 81 Topologically equivalent compartments 82 Antigen acquisition sites => TC => TH1 => TH2 83 Antigen presentation by MHC-I 84 Activation of cytotoxic T cells 85 Cytotoxic T cells induce apoptosis 86 Activation of a B cell by an antigen and TH cell 87 Antigen presentation by MHC-II 88 Summary of interplay between TH and B cells