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Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III

Core Module Immunology Doctoral Training Group GK1660 Erlangen  2011. History of Immunology Generation of Diversity - The Antibody Enigma. Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg.

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Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III

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  1. Core Module Immunology Doctoral Training Group GK1660 Erlangen  2011 History of Immunology Generation of Diversity - The Antibody Enigma Hans-Martin Jäck Division of Molecular Immunology Dept. Of Internal Medicine III Nikolaus-Fiebiger-Center University of Erlangen-Nürnberg

  2. TIME LINE - History of Immunology • Discovery of cells and germs (1683 - 1876) • Prevention of Infection (1840 – today) • Start of Immunology (1796-1910) • The antibody problem: Immunochemistry (1910 - 1975) • Self-/non-self discrimination (1940 – today) • Generation of Diversity G.O.D. (1897 and 1976s) • Discovery of B and T cells (1960s) • The molecular revolution (1976 – today)

  3. Models to Explain Immunity - Specifity & Inducibility -

  4. MODELS: Instruction versus Selection Precursor of an antibody-forming cell (AFCP) is not precommitted, but has the potential of making any one of a millions of different antibodies. AFCP) are precommitted to producing antibody of a particular specificity.

  5. Ehrlich‘s Side Chain Theory Paul Ehrlich (1854-1915) Germany Nobel price Medicine 1908 Klin Jahrb. 6:299. (1897) Proceedings of the Royal Society (London) 66, 424-448 66, 424-448

  6. 1st Selection Model (Ehrlich 1897 und 1900) Toxin Side- chain Toxin binds to specific side-chain (receptor) on cell surface like ´“a key finds ist lock Side chain-toxin complex „falls off“ from cell. Cell compensates for loss with overproduction of this side chain More specific side-chains accumulate on cell surfcae Overcrowed side-chains are released as soluble free side-chains (anti-toxin) Released antitoxins neutralize toxins Side chains (described in 1900 as “receptor”s) on the surface of cells could bind specifically to toxins – in a "lock-and-key" interaction (Emil Fischer) - and that this binding reaction was the trigger for the production of soluble antitoxins (antibodies).

  7. Key-Lock (1897) and Receptor (1900) Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299 Ehrlich & Morgenroth (1900). Über Haemolysine-dritte Mitteilung. Berliner Klinische Wochenschrift 453

  8. Emil Fischer • Synthesized (+) glucose, fructos and mannose (1890) from glycerol and purines(1898) including the first synthesis of caffeine.→ Nobel Prize for Chemistry in 1902 • 1884 (in Erlangen), coined the name “purins” for a class of active substances (caffeine and theobromine) in tea, coffee, cocoa • Discovered proline and hydroxyproline • 1890"Lock and Key Model" to explain the substrate and enzyme interaction. Emil Fischer 1852-1919 Germany Nobel Prize Chemistry 1902. Erlangen (1881-88)

  9. Emil Fischer • 1891 Fischer projections • two-dimensional representation of a three-dimensional organic molecule by projection • originally proposed for the depiction of carbohydrates and • 1901 Synthesis of the first dipeptide glycylglycine (with Ernest Fourneau) • 1919 Commits suicide (as his 2. son) In Berlin

  10. Explanation of various antibody activities • Lysine • Agglutinine • Antitoxine http://www.imedo.de/medizinlexikon/ehrlich-seitenkettentheorie

  11. Ehrlich‘s Summary: Side-Chain Theory Ehrlich (1908). Über Antigene und Antikörper. Einleitung in „Handbuch der Immunitätsforschung“. P.1 -10 Very nice overview about the knowledge of antibody and antigen in 1908.

  12. Summary: Side-Chain Theory A • Keypoints • All cells express on theirsurfacesidechains (receptors) that bind toxin. Side chains‘ physiologicfunctionistototakeupfood. (A ff) • Cell overproducesthepartcularsidechain (B) andreleasesitintothebloodstream (C) • Soluble sidechainneutralisestoxin (C), orrecruitscomplement (D), agglutinatespathogens (D asmembrane-bound form) orevenopsonises pathogen (activityonlyknownsince 1905) • Explains • all oberservedactivitiesof antibodies (agglutinins, lysins, antitoxinsandprecipitinsandevenopsonins) • Inducibility (onlypresent in bloodis soluble form after immunisation) • Specificity (onlyantibodestoparticular pathogen) • Problem • Enoughspace on a cell for all possible „toxins“ andpathogens? B C D E

  13. Landsteiner: Hapten Carrier Concept • Antibodies can be produced againts any small organic compounds and even arsenate, but only if they are coupled to protein carrier • Hapten alone does not induce antibodies but it will bind to antibodies • Antigenicity ↔ Immunogenicity

  14. Landsteiner: Antibodies against Haptens Serum derived from immunization with 3-aminobenzenesulfonic acid exhibits Landsteiner K. Die Spezifizitat Der Serologischen Reaktionen. Springer-Vertag:Berlin, 1933. Landsteiner, K. The Specificity of Serological Reactions; Harvard University Press: Cambridge, Massachusetts, 1945, p 169. (Original experiments were performed in the 20s)

  15. Landsteiner: Antibodies againts Enantiomers Landsteiner K. Die Spezifizitat Der Serologischen Reaktionen. Springer-Vertag:Berlin, 1933. Landsteiner, K. The Specificity of Serological Reactions; Harvard University Press: Cambridge, Massachusetts, 1945, p 169. (Original experiments were performed in the 20s)

  16. Landsteiner‘s Conclusion: Since he could get antibodies to arsenate as well as many other chemical groups coupled to proteins, Landsteiner reasoned that: Aus: GOLUB & Green: Immunology, a Synthesis, 2nd edition,Sunderland, MA, USA, S. 7-17

  17. Haurowitz 1930: Paradigm change p. 8 Antigen must instruct formation of specific antibody Since Landsteiner‘s work (1920s) demonstrated that antibodies can be raised against many substances that do not occur in living organisms, Ehrlich‘s side chain theory fell in disfavor and was forgotten between 1920 and 1930

  18. Models to Explain Immunity - Instructional Theories -

  19. Aera of Instructionalists Rather Antigen must instruct !!!!!! Instructionalists • Precursor of an antibody-forming cell (AFCP) is not precommitted, but has the potential of making any one of a million different antibodies. • Every precursor cell can potentially respond to any antigen.

  20. OneExampleofInstruction Gecko am Glass klebend • Each foot: half-a-million tiny hairs on the end • Each of these hairs has several hundred smaller hairs (about 0.2-0.5 microns across—same size as a wavelength of light) • Adapts to each surface Gecko Vorlesung: M. Wabl (www.herbstschule.de)

  21. Breinl‘s & Haurowitz‘ Template Theory (1930) • Antigen is taken up by special cells and serves as a template for complementary amino acids encased in the antigen • A non-specific enzyme catalyes the peptide bonds between the "complementary" amino acids. • Problems • No mechanism was described to control the size of the antibody • Could not explain the higher affinity during the 2° immunizations From K. Knight Chicago Zeitschrift Phys. Chemie 192:45, 1930

  22. Pauling‘s Template Theory (1940) • Problems • Each of the bi-valent sites could have a different binding site • The antigen needs to be present for a long time in order to “instruct” enough antibody; however, there are antibodies long after Ag has been cleared • Does not explain self/non-self discrimination

  23. Models to Explain Immunity - The Death of Instruction Theory -

  24. Primary AA Sequence Determines Structure PNAS 47 (9):1309 (1961) Nobel Price for his work on ribonuclease and specially for elucidating the correlation between amino acid sequence and biological active con-formation Christian Boehmer Anfinsen (1916 – 1995) USA Nobel Price Chemistry 1972

  25. Refolding of Ag-specific Fab (Tanford 1963) PNAS VOL. 50:827 (1963) Charles Tanford 1921 – 2009 USA

  26. Refolding of Ag-specific Fab (Haber 1964) PNAS 52:1099 (1964) Edgar Haber 1932 - 1997 USA § Separate anti-RNAse + 125I-RNAse complexes by Sephadex G-100

  27. Models to Explain Immunity - Selection Theories (Part 2) -

  28. CELLULAR SELECTION THEORIES 1955 Niels JERNE (natural selection theory) 1957 David TALMAGE (receptors should be cellular) 1957 MacFarlane BURNET (clonal selection theory) Jerne, N. K. 1955. The natural-selection theory of antibody formation. Proc. Natl. Acad. Sci. USA 41: 849–857. Talmage, D. W. 1957. Allergy and immunology. Annu. Rev. Med. 8: 239–257. Burnet, F. M. 1957. A modification of Jerne’s theory of antibody production using the concept of clonal selection. Aust. J. Sci. 20: 67–68. All rediscovered Paul Ehlrich‘s sidechain theory

  29. Natural Selection Theory (Jerne – 1955) “The "natural-selection" theory, proposed in the present paper, may be stated as follows: The role of the antigen is neither that of a template nor that of an enzyme modifier. The antigen is solely a selective carrier of spontaneously circulating antibody to a system of cells which can reproduce this antibody” Jerne, N. K. 1955. The natural-selection theory of antibody formation. Proc. Natl. Acad. Sci. USA 41: 849–857. • Minute amounts of natural Abs are present in serum (e.g., neutralizing phage Abs) • Ag forms with cognate Ab a complex, which will be phagycytosed • Phagocytosis induces production of secretable Ab • Problem: Ab-secreting cells do not phagocytose

  30. Talmage (1957) • Suggests to place antibody into a cell • Mentions Ehrlich’s work (Jerne did not) • Talmage, D. W. 1957. Allergyandimmunology. Annu. Rev. Med. 8: 239–257.

  31. Burnet (1957) The Clonal Selection Theory Burnet, F. M. 1957. A modification of Jerne’s theory of antibody production using the concept of clonal selection. Aust. J. Sci. 20: 67–68.

  32. Clonal Selection Theory (Burnet – 1957) Antigen (Antikörper generierend) Memory B cell Clonal Selection Theory (Burnet ) (1957) B cell receptor Antibody Plasma cell Monospecific B cells differentiation clonal expansion • Each AFCP is pre-committed to produce one antibody (monospecific) • Each AFCP carrys membrane-bound immunoglobulin • B cell that binds Ag gets expanded and differentiates into AFC • Explains - Specifity • Induciblity • Secondary response • Tolerance to self-antigens (clonal deletion, 1949)

  33. Selection Theory (Burnet 1957 and Ehrlich 1897) Antigen (Antikörper generierend) Memory B cell Clonal Selection Theory (Burnet ) (1957) B cell receptor Antibody Plasma cell Monospecific B cells differentiation clonal expansion Toxin Side- chain Sidechain Theory (Ehrlich 1897) Toxin binds to specific Side-chain on cell surface Binding enhances production of toxin-specific side-chains Overcrowed side-chains are released as soluble side-chains (anti-toxin) Side-chains accumulate On cell surfcae

  34. Burnet Simplified (bacterial genetics)

  35. Clonal Selection Theory: Predictions Prediction 1: One B cell should produce one kind of antibody Prediction 2: Sequences of antibodies should be different Prediction 3: Membrane bound immunoglobulin

  36. Burnet‘s Theory: Predictions Prediction 1: One B cell should produce one kind of antibody Nossal and Lederbergas well as White show that single cells from rat lymph nodes, simultaneously stimulated with two antigens, formed antibody to one or other antigen but never to both. Poly III OVA Poly III OVA • Nossal GJ, Lederberg J. Antibody production bysingle cells. Nature. 1958;181:1419-1420. • White, R. G. 1958. Antibody production by single cells. Nature 182: 1383–1384. • Nossal GJ. One cell-one antibody: prelude and aftermath. Nat Immunol. 2007;8:1015-1017. • Viret (2009). Comment on Nossal Paper. Immunol 182;1229-1230. • Spleen sections • Stain with FITC-III • Photobleach • Stain with FITC-OVA White, R. G. 1958. Nature

  37. VL CL L-Kette H chain Burnet‘s Theory: Discovery of V regions Prediction 2: Sequences of antibodies should be different Hilschmann & Craig isolated Bence Jones (L chain) from urine of three myeloma patients and found by protein sequencing that the proteins differ at the N-terminal part and are identical at the C-terminal part – V and C regions were discovered HILSCHMANN, H & LYMAN C. (1965). AMINO ACID SEQUENCE STUDIES WITH BENCE-JONES PROTEINS, PNAS 53:1403

  38. Burnet‘s Theory: Surface Immunoglobulin Prediction 3: Ig should be detected on the cell surface The authors stimulated the proliferation of rabbit spleen cells with an anti-allo-Ig antibody Sell, S. et al. (1965). STUDIES ON RABBITLYMPHOCYTES IN VITRO I. STIMITLATION OF BLAST TRANSFORMATION WITH AN ANTIALLOTYPE SERUM. JEM, p. 423

  39. Models to Explain Immunity - Genetic models -

  40. Germline Theory (e.g., Niels Jerne) V1 C V2 C V4 C V3 C Genetic Models

  41. Minimal site of a peptide epitope Number of amino acids 206 = 6 x 107 linear peptide epitopes  6 x 107 different antibodies Size of the antibody repertoire? How many different antibodies are needed?

  42. Problems – Germline Models • 1. Information for billions of antibodes can not be stored in the human genome • 20 amino acids and epitope with 6 amino acids yiels in about 206 = 6 x 107 linear epitopes • L chain: ~ 600 bases; H chain: minimal ~ 1200 bases together ~ 2000 bases • Storage space for 6 x 107 antibodies • 6x107 x 2000 = 1.2 x 1011 bases • However, human haploid genome consists of about 3 x 109 bases • 2. How is transcription of a single antibody gene regulated? • 3. How does affinity maturation work?

  43. Germline Theory (e.g., Niels Jerne) V1 C V2 C V4 C V3 C Somatic Variation Theory (e.g., Lederberg) V2a C V1 C V2 C V1 C Genetic Models

  44. Lederberg‘s Propositions (1959) Lederberg (1959). Genes and Antibodies. Science, June 1649 The stereospecific segment of each antibody globulin is determined by a unique sequence of amino acids. The cell making a given antibody has a correspondingly unique sequence of nucleotides in a segment of its chromosomal DNA: its "gene for globulinsynthesis." The genetic diversity of the precursors of antibody-forming cells arises from a high rate of spontaneous mutation during their lifelong proliferation. This hypermutability consists of the random assembly of the DNA of the globulin gene during certain stages of cellular proliferation. Each cell, as it begins to mature, spontaneously produces small amounts of the antibody corresponding to its own genotype.

  45. Lederberg‘s Propositions (1959) Lederberg (1959). Genes and Antibodies. Science, June 1649 The immature antibody-forming cell is hypersensitive to an antigen-antibody combination: it will be suppressed if it encounters the homologous antigen at this time. The mature antibody-forming cell is reactive to an antigen-antibody combination: it will be stimulated if it first encounters the homologous antigen at this time. The stimulation comprises the acceleration of protein synthesis and the cytological maturation which mark a "plasma cell.“ Mature cells proliferate extensively under antigenic stimulation but are genetically stable and therefore generate large clones genotypically preadapted to produce the homologous antibody. These clones tend to persist after the disappearance of the antigen, retaining their capacity to react promptly to its later reintroduction.

  46. Germline Theory (e.g., Niels Jerne) V1 C V2 C V4 C V3 C Somatic Variation Theory (e.g., Lederberg) V2a C V1 C V2 C V1 C Recombination Theory [Dreyer and Bennett Modell (1965)] V1 V2 V3 V4 C V1 C Genetic Models

  47. Dreyer & Bennet Recombination (1965) • Gene segments encoding the variability of the antibodies would combine with the “common" gene in antibody producing eells. • Resolves the variable/constant region paradox • UtiliIes a mechanism previously described in bacteria • Allows for generation of a highly diverse population of antibodies . • ProblemsViolates 1 gene 1 polypeptide dogma Dreyer and Benett. PNAS (1965).

  48. Brenner & Milstein Mutation Model (1961) • They proposed a model in which a 5' region of the antibody gene is degraded and error prene polymerase fills in the missing nucleotides resulting in a region highly varied sequence. • Follows nt excision repair mechanism • Allowsfor allotype maintenance • Problems • High probabiljty of non productive antibody coding sequence • Assumes timed expression of a novel error DNA polymerase Brenner & Milstein (1951) Nature

  49. Capra & Kindt (1975) – Recombination in cis Model was deduced from the discovery of the “Todd Phenomenon” - that rabbit allotypes, which were thought to be encoded by V regions, were shared by at least two if not three Ig classes (about 1963)

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