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
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 -
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.
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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
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1st Selection Model (Ehrlich 1897 und 1900)
Sidechain
Toxin
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 sidechains are released
as soluble free sidechains (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).
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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
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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
Emil Fischer
1852-1919
Germany
Nobel Prize
Chemistry
1902.
• Discovered proline and hydroxyproline
• 1890 "Lock and Key Model" to explain the
substrate and enzyme interaction.
Erlangen (1881-88)
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Emil Fischer
• 1891 Fischer projections
o two-dimensional representation of a three-dimensional
organic molecule by projection
o originally proposed for the depiction of carbohydrates
and
In Berlin
• 1901 Synthesis of the first dipeptide glycylglycine
(with Ernest Fourneau)
• 1919 Commits suicide (as his 2. son)
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Explanation of various antibody activities
o Lysine
o Agglutinine
o Antitoxine
http://www.imedo.de/medizinlexikon/ehrlich-seitenkettentheorie
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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.
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Summary: Side-Chain Theory
A
B
D
Keypoints
C
E
o
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)
Problem
o
Enough space on a cell for all possible „toxins“ and pathogens?
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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
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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)
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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)
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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
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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
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Models to Explain Immunity
- Instructional Theories -
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.
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One Example of Instruction
Gecko am Glass
klebend
o Each foot: half-a-million tiny hairs on the end
Gecko
o Each of these hairs has several hundred smaller hairs
(about 0.2-0.5 microns across—same size as a
wavelength of light)
o Adapts to each surface
Vorlesung: M. Wabl (www.herbstschule.de)
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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
From K. Knight
Chicago
• Could not explain the higher
affinity during the 2°
immunizations
Zeitschrift Phys. Chemie 192:45, 1930
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Pauling‘s Template Theory (1940)
Problems
o 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/nonself discrimination
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Models to Explain Immunity
- The Death of Instruction Theory -
Primary AA Sequence Determines Structure
PNAS 47 (9):1309 (1961)
Christian
Boehmer
Anfinsen
(1916 – 1995)
USA
Nobel Price for his work
on ribonuclease and
specially for elucidating
the correlation between
amino acid sequence
and biological active
con-formation
Nobel Price
Chemistry
1972
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Refolding of Ag-specific Fab (Tanford 1963)
PNAS VOL. 50:827 (1963)
Charles
Tanford
1921 – 2009
USA
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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
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Models to Explain Immunity
- Selection Theories (Part 2) -
CELLULAR SELECTION THEORIES
1955 Niels JERNE (natural selection theory)
Jerne, N. K. 1955. The natural-selection theory of antibody formation. Proc. Natl. Acad. Sci.
USA 41: 849–857.
1957 David TALMAGE (receptors should be cellular)
Talmage, D. W. 1957. Allergy and immunology. Annu. Rev. Med. 8: 239–
257.
1957 MacFarlane BURNET (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.
All rediscovered Paul Ehlrich‘s sidechain theory
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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.
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
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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.
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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.
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Clonal Selection Theory (Burnet – 1957)
Clonal
Selection
Theory
(Burnet )
(1957)
Antigen
(Antikörper
generierend)
Memory
B cell
B cell
receptor
Antibody
Plasma
cell
Monospecific
B cells
clonal expansion
o
o
o
o
differentiation
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)
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Selection Theory (Burnet 1957 and Ehrlich 1897)
Clonal
Selection
Theory
(Burnet )
(1957)
Antigen
(Antikörper
generierend)
Memory
B cell
B cell
receptor
Antibody
Plasma
cell
Monospecific
B cells
clonal expansion
Sidechain
Sidechain
Theory
(Ehrlich 1897)
differentiation
Toxin
Toxin binds
to specific
Side-chain on
cell surface
Binding enhances
production of
toxin-specific
side-chains
Side-chains
accumulate
On cell surfcae
Division of Molecular Immunology, Universitätsklinikum Erlangen
Overcrowed sidechains are released as
soluble side-chains
(anti-toxin)
56
Burnet Simplified (bacterial genetics)
Division of Molecular Immunology, Universitätsklinikum Erlangen
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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
Division of Molecular Immunology, Universitätsklinikum Erlangen
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Burnet‘s Theory: Predictions
Prediction 1: One B cell should produce one kind of antibody
Nossal 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.
Poly III OVA
Poly III
OVA
o Nossal GJ, Lederberg J.
Antibody production bysingle
cells. Nature. 1958;181:14191420.
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.
o
o
o
o
Spleen sections
Stain with FITC-III
Photobleach
Stain with FITC-OVA
White, R. G. 1958. Nature
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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
VL
L-Kette
CL
H chain
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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 RABBIT LYMPHOCYTES IN VITRO I. STIMITLATION OF BLAST TRANSFORMATION WITH AN
ANTIALLOTYPE SERUM. JEM, p. 423
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Models to Explain Immunity
- Genetic models -
Genetic Models
Germline Theory (e.g., Niels Jerne)
V1
C
V2
C
V3
C
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V4
C
82
Size of the antibody repertoire?
How many different antibodies are needed?
Number of
amino acids
Minimal site of a
peptide epitope
206 = 6 x 107 linear peptide epitopes
 6 x 107 different antibodies
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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?
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Genetic Models
Germline Theory (e.g., Niels Jerne)
V1
C
V2
C
V3
V4
C
C
Somatic Variation Theory (e.g., Lederberg)
V1
C
V2
C
V1
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C
V2a
C
86
Lederberg‘s Propositions (1959)
Lederberg (1959). Genes and Antibodies. Science, June 1649
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.
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Lederberg‘s Propositions (1959)
Lederberg (1959). Genes and Antibodies. Science, June 1649
6. The immature antibody-forming cell is hypersensitive to an antigenantibody 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.
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Genetic Models
Germline Theory (e.g., Niels Jerne)
V1
C
V2
C
V3
V4
C
C
Somatic Variation Theory (e.g., Lederberg)
V1
C
V2
C
V1
C
V2a
C
Recombination Theory [Dreyer and Bennett Modell (1965)]
V1
V2
V3
V4
C
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V1
C
90
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 Problems
Violates 1 gene 1 polypeptide dogma
Dreyer and Benett. PNAS (1965).
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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
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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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Todd‘s Phenomenon
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Tonegawa (1976)
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The Tonegawa Experiment(1976)
32P
Hybridize
Cut in gel pieces
Elute and
denature DNA
Radioactivity
in dsDNA
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Somatic Recombination → The Key Experiment
The explanation
Myeloma
DNA
Liver
DNA
The experiment
Germline
E
8kb
S. Tonegawa
Nobel Price 1987
Basel Institute
of Immunology
6kb
E
E
E
4kb
6kb
V
C
probe
probe
4kb
recombination
Myeloma
Probe
E
E
8kb
(radiolabelled L chain mRNA)
V
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C
102
The Direct Prove (1977)
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 changes
in the content and context of immunoglobulin genes. Cold Spring Harbor Symp.
Quant. Biol. 41:877.
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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.
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Discovery of Recombination Signals (1978)
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Discovery of D segments (1980)
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Discovery of Recombination Enzymes
• 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.
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Etablishment of primary V repertoire

13 DH

4 JH

5 CH
VH regions
(ca. 6760)
Recombinatorial
diversity

~ 85 Vκ

4 Jκ
Vκ regions
(340)

1 Cκ
Recombinatorial
diversität
(reperire, lat.
wiederfinden)
Combinatorial Diversity
 ~ 134 VH
mouse
Doctoral Training Group GK1660 - University of Erlangen-Nürnberg
~ 2,3x107 Abs
V(D)J recombination
generates
antibody diversity
113
Size of the antibody repertoire?
How many different antibodies are needed?
Number of
amino acids
Minimal site of a
peptide epitope
206 = 6 x 107 linear peptide epitopes
 6 x 107 different antibodies
Doctoral Training Group GK1660 - University of Erlangen-Nürnberg
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Verknüpfungsdiversität 1+2 (N and P nucleotid addition)
134 VH
4JH
13 D
C
Rag1/2
Random processing of hairpin and insertion of non-templated nucleotides
P, palindromic
N
P
a
b
C-A-C
G-T-G
a
• Ku70/80
• DNA-PK
• Artemis
CG-T-G-C-A
C-A-C-G-T -N-N-N
G-T-G-C-A -N-N-N
• Pol
b
C-A-C
G-T-G
• TdT
Junctional
diversity
C-A-C -N-N-N
G-T-G -N-N-N
Junctional diversity increases antibody repertoire
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Verknüpfungsdiversität 3 (Junctional Diversity)
D-Segmente können in allen 3 Leseraster benützt werden durch
V
D
..GGG AAA CCT
TTAGTCACATTCCCG
J
ACG
AAA TTT ....
AGTCACATTCCC
Nonsense
Codon
TAGTCACATTCC
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Etablishment of primary V repertoire (mouse)

13 DH

4 JH

5 CH
VH regions
(ca. 6760)
Recombinatorial
diversity

~ 85 Vκ

4 Jκ
Vκ regions
(340)

1 Cκ
Recombinatorial
diversität
(reperire, lat.
wiederfinden)
Combinatorial Diversity
 ~ 134 VH
mouse
Doctoral Training Group GK1660 - University of Erlangen-Nürnberg
~ 2,3x107 Abs
Junctional
diversity
109 - 1012 Abs
117
Summary: Preimmune Repertoire
134 VH
13 D
4JH
C
Recombinatorial diversity
• Random assembly from V, D & J
Combinatorial diversity
ca. 107
antibodies
• Random pairing of H & L chains
Junctional diversity
109-1012
antibodies
• Unprecise V(D)J joining
• Nucleotide (N) addition (TdT)
• Usage of three RF in D segments
Doctoral Training Group GK1660 - University of Erlangen-Nürnberg
118