ab diversity ppt.pptjbvjgcxgxghchjvknlnbklnkln

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1. Immunological diversity

2. 
Human preimmune repertoire needs a
capacity of ~ 1015 to cope with antigens
encountered in life time.

Human genome has ~ 105 genes, not enough
to code for all in preimmune repertoire.
Antibody Diversity

3. Clonal selection
•– Each B cell makes only one antibody and is a
clone (each cell has ~105 immunoglobulin
molecules on the surface).
•The binding of antigen to the receptor on the surface
of naïve cell (when the cell is in the peripheral
lymphoid organ)activates the B cell clone to develop
into effector cells and to proliferate

4. Clonal selection

5. Clonal selection

After the first exposure, some become memory
cells.

Both memory and naïve B cells are activated by
antigen binding.

Memory cells respond differently than naïve cells,
resulting in a higher antibody titer

6. Clonal selection

7. Immunoglobulins or Antibodies
• Classes of antibodies (differ in their heavy chain)
• 5 classes of heavy chain IgA, IgD, IgE, IgG, IgM with α, δ,
ε, γ, μ heavy chain respectively IgG, IgA have subclasses
(IgG1, IgG3, IgG3, IgG4 with γ1, γ2, γ3, γ4 respectively).
• 2 light chain κ or λ
• Constant region and variable region
• Both heavy chain and light chain have a constant region
and a variable region.
•Three hypervariable section are the loci which are really
variable in the variable region of heavy and light chains,
the rest of “framework” in the variable region is relatively
invariant.

8. The antibody molecule

9. Antibody diversity is generated by
a number of mechanisms
• Combinational recombination
Mammal recombine immunoglobulin gene segments before
transcription.
•Each chain (λ, κ, and heavy chains) has a pool of gene segments.
• Each pool is on a different chromosome. i.e. on one chromosome.
• There are a large pool of segments for variable region and a
smaller pool of constant region.
• During B cell development, a coding sequence is assembled by site
specific recombination to increase the diversity. An antibody IgD
with a К light chain thus has 7 x 106 possible combination.

10. Each variable region of a light chain is made
of a V and a J gene segment

11. Human heavy chain gene segment
pool
•Each variable region of a heavy chain is
madeof a, V, J, D segment

12. Imprecise V(D)J joining
•Only appropriate segments get joined (V always
joins to J in a light chain, not V to V). It is site
specific.
• Catalysed by RAG-1 and RAG-2 (recombination
activating genes)
• During recombination, nucleotides can be lost or
inserted (may shift reading frame)
• Can have a second round of V(D)J rearrangement
(receptor editing)

13. Mouse immunoglobulin genes
V1-V500D1-D12 J1-J4
H chain locus (Chr 12)
CCC3C1
C2bC2aCC
V1-V250 J1-J5 C
V2 J2C2
 chain locus (Chr 6)
 chain locus (Chr 16)
C3 C1V1 J3 J1

14. Mechanisms for generating
antibody diversity

V(D)J recombination

Somatic hypermutation

Class switch recombination
V1-V500 D1-D12 J1-J4 constant regions

15. 
V(D)J recombination
Mechanisms for generating
antibody diversity
V1-V500 D1-D12 J1-J4 constant regions
Hozumi and Tonegawa, PNAS 1976

16. 
V(D)J recombination
Mechanisms for generating
antibody diversity
V1-V500 D1-D12 J1-J4 constant regions

17. 
V(D)J recombination
Mechanisms for generating
antibody diversity
V1-V500 D1-D12 J1-J4 constant regions

18. 
V(D)J recombination
D to J joining
Mechanisms for generating
antibody diversity

19. 
V(D)J recombination
D to J joining
Mechanisms for generating
antibody diversity

20. 
V(D)J recombination
V to DJ joining
Mechanisms for generating
antibody diversity

21. 
V(D)J recombination

Somatic hypermutation
** *
Somatic
mutations
Mechanisms for generating
antibody diversity

22. ** *

V(D)J recombination

Somatic hypermutation

Class switch recombination
Class switch
Mechanisms for generating
antibody diversity

23. 
V(D)J recombination

Somatic hypermutation

Class switch recombination
** *
Class switch
Mechanisms for generating
antibody diversity

24. 
V(D)J recombination

Somatic hypermutation

Class switch recombination
** *
V(D)J recombination and class switch recombination
involve double-strand breaks
Mechanisms for generating
antibody diversity

25. Recombination signal sequence (RSS)
direct V(D)J recombination
23
CACAGTG–––––––ACAAAAACC
23
GGTTTTTGT–––CACTGTG
12
heptamer nonamernonamer heptamer
12
V

J

The 12/23 rule: recombination occurs between
a RSS with a 12 bp spacer and a RSS with a 23 bp spacer

26. V(D)J recombination involves DNA
cleavage and end-joining
coding joinsignal join
cleavage
end-joining

27. Cleavage is initiated by RAG1/RAG2
(recombination activating genes)
RAG1/RAG2 nick DNA at two RSSs....
then catalyze nucleophilic attack by 3' OH on the opposite strand
5' OH
5' OH
5'
5'
generating hairpin coding ends and blunt signal ends
van Gent, Gellert et al. Cell 1995

28. DNA ends are modified by
addition and deletion

N-nucleotide addition by terminal
deoxynucleotidyl transferase (TdT)

P-nucleotide addition by asymmetric
opening of hairpin coding ends

Nucleotide deletion

29. Addition at DNA ends
TCA
AGT
TCA
AGT
N-nucleotide addition
addition at 3' ends by TdT
addition
Germ-line gene Rearranged gene Mechanism
GATCA
CTAGT
P-nucleotide addition
hairpin formation and
asymmetric cleavage
addition
TCA
AGT
GATCA
T
ATAGT
TATCA

30. Deletion at DNA ends
.
Germ-line gene Rearranged gene Mechanism
CA
GT
TCA
AGT
Nucleotide deletion
random
deletion
TCA
AGT
Nucleotide deletion
microhomology directed
deletion
TCA
AGT
CA
GT
T
C
A
G
TC
A
G
T

31. DNA pathways in V(D)J recombination

32. Evolution of V(D)J recombination

RAG1 and RAG2 contain no introns and
are tightly linked on the same chromosome

RAG1 and RAG2 are conserved back to
the evolution of jawed fish

Evolutionary hypothesis: a transposon
with RAG1, RAG2, and associated RSSs
infected a precursor of jawed fish

33. RAG1 and RAG2 do not exist
in jawless fish
hagfishlamprey

34. Hypothetical RAG transposon
RAG1 RAG2
excision

35. Transposon integration
5' 5'
5'
5' 5'
5'
OH OH
Agrawal, Eastman and Schatz Nature 1998

36. Origin of the immunoglobulin genes
D JV
ancient receptor gene
V
gene duplication
D12 J4V500 C
first transposon integration
second transposon integration

37. Mechanisms for repairing
DNA double-strand breaks

Homologous recombination

Non-homologous end-joining
V(D)J recombination mutants are defective
in non-homologous end-joining

38. Proteins involved in
non-homologous end-joining
Protein Enzymatic activity
Ku DNA end-binding
DNA-PKcs DNA-dependent protein kinase
XRCC4/ Ligase 4 DNA ligase
Artemis exonuclease
Rad50/ Mre11/ Nbs1 exo/endonuclease

39. Somatic hypermutation (SHM)

SHM targets immunoglobulin genes
(but not T cell receptor genes)

SHM requires active transcription

SHM involves DNA single-strand breaks

40. Model for somatic hypermutation

Activation-induced deaminase (AID)

Expressed only in activated B cells

Converts C to U in single-stranded DNA

Other proteins insert mutations

Uracil DNA glycosylase converts U to an apurinic site

AP endonuclease nicks the DNA adjacent to the AP site

Exonuclease removes the AP ribose

An error-prone polymerase fills in the gap

41. Model for somatic hypermutation
How is C mutated on both strands with the same frequency?
How does SHM target the Ig locus, but not other loci?

42. Class switch recombination (CSR)

CSR rearranges the constant regions to
generate different antibody isotypes

CSR regions
located 5’ to each C
H gene, except for C


consist of repeats of GAGCT and GGGGGT;
e.g.,  switch region is [(GAGCT)
nGGGGGT]
150

CSR requires active transcription

AID initiates CSR

43. CSR occurs via double-strand breaks

CSR requires Ku and DNA-PKcs

CSR junctions show characteristics of
non-homologous end-joining

Deletions to regions of microhomology

Duplications from DNA polymerase activity

44. Model for class switch recombination
How does AID initiate CSR at one locus and SHM at another?
(The C-terminus of AID is required for CSR, but not SHM.)