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

Hans-Martin JäckDivision of Molecular ImmunologyDept. Of Internal Medicine IIINikolaus-Fiebiger-CenterUniversity of Erlangen-Nürnberg

History of ImmunologyGeneration of Diversity - The Antibody Enigma

Core Module ImmunologyDoctoral Training Group GK1660Erlangen 2011

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)

Models to Explain Immunity- Specifity & Inducibility -

Doctoral Training Group GK1660 - University of Erlangen-Nürnberg 5

AFCP) are precommitted to producing antibody of a particular specificity.

Precursor of an antibody-forming cell (AFCP) is not precommitted, but has the potential of making any one of a millions of different antibodies.

MODELS: Instruction versus Selection


Ehrlich‘s Side Chain Theory

Klin Jahrb. 6:299. (1897)

Proceedings of the Royal Society (London) 66, 424-448 66, 424-448

Paul Ehrlich(1854-1915)

GermanyNobel price Medicine

1908


1st Selection Model (Ehrlich 1897 und 1900)

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).

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)

Toxin binds to specific side-chain

(receptor) on cell surface

like ´“a key finds ist lock

ToxinSide-chain

Released antitoxins neutralize

toxins


Ehrlich, P (1897). Wertbemessung des Diphterieheilserums - Grundlagen. Klin Jahrb. 6:299

Ehrlich & Morgenroth (1900). Über Haemolysine-dritte Mitteilung. Berliner Klinische Wochenschrift 453

Key-Lock (1897) and Receptor (1900)


Emil Fischer

Emil Fischer 1852-1919Germany

Nobel Prize Chemistry

1902.

Erlangen (1881-88)

• 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.


• 1891 Fischer projections o two-dimensional representation of a three-dimensional

organic molecule by projectiono 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)

Emil Fischer

In Berlin


http://www.imedo.de/medizinlexikon/ehrlich-seitenkettentheorie

Explanation of various antibody activities

o Lysine

o Agglutinine

o Antitoxine


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.

Ehrlich‘s Summary: Side-Chain Theory


Keypointso All cells express on their surface sidechains (receptors) that bind

toxin. Side chains‘ physiologic function is to to take up food. (A ff)

o Cell overproduces the partcular sidechain (B) and releases it into the bloodstream (C)

o Soluble sidechain neutralises toxin (C), or recruits complement (D), agglutinates pathogens (D as membrane-bound form) or even opsonises pathogen (activity only known since 1905)

Explains o all oberserved activities of antibodies (agglutinins, lysins,

antitoxins and precipitins and even opsonins)

o Inducibility (only present in blood is soluble form after immunisation)

o Specificity (only antibodes to particular pathogen)

Problemo Enough space on a cell for all possible „toxins“ and pathogens?

A

B C

D E

Summary: Side-Chain Theory


• 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

Landsteiner: Hapten Carrier Concept


Landsteiner: Antibodies against Haptens

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)

Serum derived from immunization with 3-aminobenzenesulfonic acid exhibits


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)


Aus: GOLUB & Green: Immunology, a Synthesis, 2nd edition,Sunderland, MA, USA, S. 7-17

Since he could get antibodies to arsenate as well as many other chemical groups coupled to proteins, Landsteiner reasoned that:

Landsteiner‘s Conclusion:


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

Haurowitz 1930: Paradigm change

p. 8

Antigen must instruct formation of specific antibody

Models to Explain Immunity- Instructional Theories -


Rather Antigen must instruct !!!!!! Instructionalists

Aera of 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.


One Example of Instruction

Vorlesung: M. Wabl (www.herbstschule.de)

oEach foot: half-a-million tiny hairs on the end

oEach of these hairs has several hundred smaller hairs (about 0.2-0.5 microns across—same size as a wavelength of light)

oAdapts to each surfaceGecko

Gecko am Glass klebend


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

Zeitschrift Phys. Chemie 192:45, 1930

From K. KnightChicago


Pauling‘s Template Theory (1940)

Problemso Each of the bi-valent sites

could have a different binding site

o 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

o Does not explain self/non-self discrimination

Models to Explain Immunity- The Death of Instruction Theory -


Christian Boehmer Anfinsen (1916 – 1995)

USA

Nobel Price Chemistry

1972

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


Refolding of Ag-specific Fab (Tanford 1963)

PNAS VOL. 50:827 (1963)

Charles Tanford

1921 – 2009USA


Refolding of Ag-specific Fab (Haber 1964)

PNAS 52:1099 (1964)

Edgar Haber

1932 - 1997USA

§ Separate anti-RNAse + 125I-RNAse complexes by Sephadex G-100

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


1955 Niels JERNE (natural selection theory)

1957 David TALMAGE (receptors should be cellular)

1957 MacFarlane BURNET (clonal selection theory)

CELLULAR SELECTION THEORIES

All rediscovered Paul Ehlrich‘s sidechain 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.


“The "natural-selection" theory, proposed in the present paper, may be stated asfollows: The role of the antigen is neither that of a template nor that of an enzymemodifier. 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.

Natural Selection Theory (Jerne – 1955)

o Minute amounts of natural Abs are present in serum (e.g., neutralizing phage Abs)

o Ag forms with cognate Ab a complex, which will be phagycytosed

o Phagocytosis induces production of secretable Ab

o Problem: Ab-secreting cells do not phagocytose


Talmage (1957)

o Suggests to place antibody into a cell

o Mentions Ehrlich’s work (Jerne did not)

Talmage, D. W. 1957. Allergy and immunology. Annu. Rev. Med. 8: 239–257.


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.


Clonal Selection

Theory(Burnet )

(1957)

clonal expansion differentiation

Memory B cell

Monospecific B cells

Antibody

B cellreceptor

Antigen(Antikörper

generierend)

Clonal Selection Theory (Burnet – 1957)

o Each AFCP is pre-committed to produce one antibody (monospecific)o Each AFCP carrys membrane-bound immunoglobulino B cell that binds Ag gets expanded and differentiates into AFCo Explains - Specifity

- Induciblity

- Secondary response

- Tolerance to self-antigens (clonal deletion, 1949)

Plasma cell

Division of Molecular Immunology, Universitätsklinikum Erlangen 56

Clonal Selection

Theory(Burnet )

(1957)

clonal expansion differentiation

Memory B cell

Monospecific B cells

Antibody

B cellreceptor

Antigen(Antikörper

generierend)

Selection Theory (Burnet 1957 and Ehrlich 1897)

Plasma cell

Binding enhances production of

toxin-specific side-chains

Side-chains accumulate

On cell surfcae

Overcrowed side-chains are released

as soluble side-chains(anti-toxin)

Sidechain Theory

(Ehrlich 1897)Toxin binds to specific

Side-chain on cell surface

ToxinSide-chain


Burnet Simplified (bacterial genetics)


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


Prediction 1: One B cell should produce one kind of antibodyNossal and Lederberg as 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.

Burnet‘s Theory: Predictions

White, R. G. 1958. Nature

o Nossal GJ, Lederberg J. Antibody production bysingle cells. Nature. 1958;181:1419-1420.

o White, R. G. 1958. Antibody production by single cells. Nature 182: 1383–1384.

o Nossal GJ. One cell-one antibody: prelude and aftermath. Nat Immunol. 2007;8:1015-1017.

o Viret (2009). Comment on Nossal Paper. Immunol 182;1229-1230.

Poly III OVAPoly III OVA

o Spleen sectionso Stain with FITC-IIIo Photobleacho Stain with FITC-OVA


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

Burnet‘s Theory: Discovery of V regions

L-Kette

H chain

VL

CL


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 RABBIT LYMPHOCYTES IN VITRO I. STIMITLATION OF BLAST TRANSFORMATION WITH AN ANTIALLOTYPE SERUM. JEM, p. 423

Burnet‘s Theory: Surface Immunoglobulin

Models to Explain Immunity- Genetic models -


Germline Theory (e.g., Niels Jerne)

V1 C V2 C V3 C V4 C

Genetic Models


Size of the antibody repertoire?

206 = 6 x 107 linear peptide epitopes

6 x 107 different antibodies

Number of amino acids

Minimal site of a peptide epitope

How many different antibodies are needed?


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?

Problems – Germline Models



V1 C V2 C V3 C V4 C

Somatic Variation Theory (e.g., Lederberg)

V1 C V2 C V2a CV1 C

Genetic Models


Lederberg‘s Propositions (1959)

1. The stereospecific segment of each antibody globulin is determined by a unique sequence of amino acids.

2. The cell making a given antibody has a correspondingly unique sequence of nucleotides in a segment of its chromosomal DNA: its "gene for globulinsynthesis."

3. The genetic diversity of the precursors of antibody-forming cells arises from a high rate of spontaneous mutation during their lifelong proliferation.

4. This hypermutability consists of the random assembly of the DNA of the globulin gene during certain stages of cellular proliferation.

5. Each cell, as it begins to mature, spontaneously produces small amounts of the antibody corresponding to its own genotype.

Lederberg (1959). Genes and Antibodies. Science, June 1649


Lederberg‘s Propositions (1959)

6. 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.

7. 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.“

8. Mature cells proliferate extensively under antigenic stimulation but are genetically stable and therefore generate large clones genotypically preadapted to produce the homologous antibody.

9. These clones tend to persist after the disappearance of the antigen, retaining their capacity to react promptly to its later reintroduction.

Lederberg (1959). Genes and Antibodies. Science, June 1649



V1 C V2 C V3 C V4 C

Somatic Variation Theory (e.g., Lederberg)

V1 C V2 C V2a CV1 C

Recombination Theory [Dreyer and Bennett Modell (1965)]

V1 V2 V3 V4 C V1 C

Genetic Models


Dreyer & Bennet Recombination (1965)

o Gene segments encoding the variability of the antibodies would combine with the “common" gene in antibody producing eells.

o Resolves the variable/constant region paradox

o UtiliIes a mechanism previously described in bacteria

o Allows for generation of a highly diverse population of antibodies .

o ProblemsViolates 1 gene 1 polypeptide dogma

Dreyer and Benett. PNAS (1965).


Brenner & Milstein Mutation Model (1961)

o 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.

o Follows nt excision repair mechanism

o Allowsfor allotype maintenance

o Problems• High probabiljty of non productive antibody

coding sequence• Assumes timed expression of a novel error

DNA polymerase

Brenner & Milstein (1951) Nature


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)

Capra & Kindt (1975) – Recombination in cis


Todd‘s Phenomenon


Tonegawa (1976)


32P

Cut in gel pieces

Elute anddenature DNA

Radioactivity in dsDNA

Hybridize

The Tonegawa Experiment(1976)


Somatic Recombination → The Key Experiment

S. TonegawaNobel Price 1987Basel Institute of Immunology

Probe (radiolabelled L chain mRNA)

6kb

4kb

8kb

Liv

er

DN

A

Mye

lom

a D

NA

The experiment

6kb

8kb

probe probe

E EE

E E

The explanation

recombination

E4kb

Germline

Myeloma

V

V

C

C


The direct proof would come a year later, when at the Cold Spring Harbor Antibody meeting, Tonegawa presented his finding that V and C rearranged between embryonic and adult B cells.

Tonegawa, S., N. Hozumi, G. Matthyssens, and R. Schuller. 1977. Somatic changesin the content and context of immunoglobulin genes. Cold Spring Harbor Symp.Quant. Biol. 41:877.

The Direct Prove (1977)


Discovery of V and J segments

Within 2 years, his lab, Phil Leder’s lab, and others had decisively shown that there were “two genes per variable region” (V and J in L chains).

• Seidman, J. G., Leder, A., Edgell, M. H., Polsky, F., Tilghman, S. M., Tiemeier, D. C. & Leder, P. (1978) Proc. Natl. Acad. Sci. USA 75,3881-3885.

• Rabbitts, T. H. & Forster, A. (1978) Cell 13,319-327.

• Bernard, O., Hozumi, N. & Tonegawa, S. (1978) Cell 15, 1133-1144.


Discovery of Recombination Signals (1978)


Discovery of D segments (1980)


• Baltimore, D. 1974. Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes? Nature 248: 409–411.

• Schatz, D. G., and D. Baltimore. 1988. Stable expression of immunoglobulin gene V(D)J recombinase activity by gene transfer into 3T3 fibroblasts. Cell 53: 107–115.

• Schatz, D. G., M. A. Oettinger, and D. Baltimore. 1989. The V(D)J recombination activating gene, RAG-1. Cell 59: 1035–1048.

• Oettinger, M. A., D. G. Schatz, C. Gorka, and D. Baltimore. 1990. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248: 1517–1523.

• McBlane, J. F., D. C. van Gent, D. A. Ramsden, C. Romeo, C. A. Cuomo, M. Gellert, and M. A. Oettinger. 1995. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83: 387–395.

• van Gent, D. C., K. Mizuuchi, and M. Gellert. 1996. Similarities between initiation of V(D)J recombination and retroviral integration. Science 271: 1592–1594.

• Agrawal, A., Q. M. Eastman, and D. G. Schatz. 1998. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394: 744–751.

• Hiom, K., M. Melek, and M. Gellert. 1998. DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations. Cell 94: 463–470.

Discovery of Recombination Enzymes


~ 85 Vκ Vκ regions(340) 4 Jκ

1 Cκ

VH regions(ca. 6760) 4 JH

5 CH

~ 134 VH

13 DH

Etablishment of primary V repertoire

Recombinatorialdiversity

~ 2,3x107 Abs

Co

mb

ina

toria

l Div

ersi

ty

(reperire, lat. wiederfinden)

V(D)J recombination generates

antibody diversity

Recombinatorialdiversität

mouse


Size of the antibody repertoire?

206 = 6 x 107 linear peptide epitopes

6 x 107 different antibodies

Number of amino acids

Minimal site of a peptide epitope

How many different antibodies are needed?


4JH C134 VH 13 D

C-A-C G-T-G

C-A-C-G-T G-T-G-C-A

C-A-C G-T-G

C- G-T-G-C-A

C-A-C G-T-G

• Ku70/80• DNA-PK• Artemis

a b a

b

Junctional diversity

Random processing of hairpin

Rag1/2

Verknüpfungsdiversität 1+2 (N and P nucleotid addition)

Junctional diversity increases antibody repertoire

• TdT

-N-N-N -N-N-N

-N-N-N -N-N-N

and insertion of non-templated nucleotides

P N

P, palindromic

• Pol


..GGG AAA CCT TTAGTCACATTCCCG ACG AAA TTT ....

AGTCACATTCCC

D-Segmente können in allen 3 Leseraster benützt werden durch

Verknüpfungsdiversität 3 (Junctional Diversity)

V D J

TAGTCACATTCCNonsenseCodon


109 - 1012 Abs

Junctional diversity

~ 85 Vκ Vκ regions(340) 4 Jκ

1 Cκ

VH regions(ca. 6760) 4 JH

5 CH

~ 134 VH

13 DH

Etablishment of primary V repertoire (mouse)

Recombinatorialdiversity

~ 2,3x107 Abs

Co

mb

ina

toria

l Div

ersi

ty

(reperire, lat. wiederfinden)

Recombinatorialdiversität

mouse


4JH C134 VH 13 D

Recombinatorial diversity• Random assembly from V, D & J

Combinatorial diversity• Random pairing of H & L chains

ca. 107 anti-

bodies

109-1012 anti-

bodiesJunctional diversity• Unprecise V(D)J joining• Nucleotide (N) addition (TdT)• Usage of three RF in D segments

Summary: Preimmune Repertoire

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

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