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Origin of diversity
Bibliography
Antibody diversity: one enzyme to rule them all
Michel C Nussenzweig & Frederick W Alt
Nature Medicine 10;1304-5;2004
Immunobiology Janeway et al 6th ed 2005 (or 7th ed. 2008)
‫‪Clonal selection theory‬‬
‫יצירת מאגר של‬
‫לימפוציטים בשלים‬
‫תא אב יחיד מתמיין‬
‫למספר גדול של‬
‫לימפוציטים שלכל‬
‫אחד מהם ספציפיות‬
‫שונה‬
‫חלוקה והתמיינות‬
‫של לימפוציטים‬
‫ספציפיים ליצירת‬
‫קלון של תאים‬
‫אפקטוריים‬
‫סילוק של לימפוציטים‬
‫לא בשלים המגיבים‬
‫עם אנטיגנים עצמיים‬
‫‪Memory‬‬
‫‪cells‬‬
‫‪T cells:‬‬
‫‪T-helper T-killer‬‬
‫‪B-cells:‬‬
‫‪plasma cells‬‬
‫‪Ab secretion‬‬
Figure 3-1 part 3 of 3
Figure 3-6
CDR1 CDR2
CDR3
CDR1
CDR2
CDR3
Figure 4-2
The chance for successful rearrangement is 33%
Ontogeny of the B Lymphocyte
• Early differentiation
– Many stages defined by Ig gene
rearrangements
– Progenitor cells
•
•
•
•
Hematopoietic stem cells
Pro-B
Pre-B
Immature B
Pathway of B cell Differentiation
Antigen Independent
Cytoplasmic
m chain
Heavy chain +
surrogate light
chain )‫((חליפית‬
Antigen Dependent
“Heavy” plus
“light” chain
IgG “Memory” cell
Mature
IgM + IgD
Pre-Pro B
Pro B
D--->JH
Pre-B1
VH-->DJH
Pre-B2
B
Immature
IgM
VL-->JL
“Plasmablast”
Ontogeny of the B Lymphocyte
• Process of differentiation
–Signals that promote survival and
proliferation of early cells
• Adhesive interactions with stroma
(non-lymphoid cells that make up
marrow matrix)
• Secretion of IL7
Ontogeny of the B Lymphocyte
• Process of differentiation (continued)
– Early cells
• Pro-B – heavy chain D gene segment
rearranges to J segment
• Pre-B – Heavy chain V gene segment
rearranges to join DJ region
– Rearranged VDJ put close to heavy chain
constant m gene
– Synthesizes a m heavy chain
Figure 7-4 part 1 of 3
D-J
rearrangement
V-DJ
rearrangement
Figure 4-2
Figure 7-6 part 1 of 2
DJ rearrangement on both chromosomes
Ontogeny of the B Lymphocyte
• Pre-B cell – expresses mchain as a trans-membrane
molecule at cell surface
– In conjunction with products of two non-rearranged genes
• Lambda 5 and VpreB
– Function as surrogate light chains
– Also in conjunction with additional trans-membrane molecules
linked by disulfides
• Ig alpha (CD79a)
• Ig beta (CD79b)
– Combination of tran-smembrane molecules (pre-B cell receptor;
pre-BCR)
m heavy chain with
surrogate light chains
IL-7R (IL-7Ra and IL-2 gc chain)
Iga and Igb proteins
IL-7 and adhesive interactions
between B cells and stromal
cells important for proliferation
l5 KO blocks L chain rearrangement
and B cell differentiation
It does not block second H-chain
rearrangement
Signal Transduction;
1. clonal expansion
2. allelic exclusion
3. light chain rearrangement
Figure 7-18
Sensitive to self antigens
Figure 7-6 part 2 of 2
Sensitive to self
antigens
Allelic exclusion
Ontogeny of the B Lymphocyte
• Immature B cells
– L chains pair with mH chains to form monomeric IgM
then inserted into membrane
– Recognize Ag and respond to it
• Long-lasting inactivation instead of expansion and
differentiation
• Interact with self-Ag in bone marrow – inactivate cell
– Called negative selection
– Important for development of self-tolerance in B-lineage
» Cells with potential reactivity to self prevented from
responding (central tolerance)
Ontogeny of the B Lymphocyte
• Development of self-tolerance
– Immature B cell exposed in bone marrow to:
• Self-molecule on surface cells
– Apoptosis (deletion)
• Non-cell surface molecule (soluble Ag)
– Cell is inactivated but not deleted (anergized)
• Reactivation of VDJ recombinase (receptor editing)
– Ig L chain genes undergo secondary rearrangement
» Use unrearranged V or J segments
» Generates specificity for non-self Ag – rescued from
inactivation
After gene rearrangements and production of a functional
molecule, the cell tests whether its specificity is anti-self
Maintenance of
tolerance requires the
persistence of antigen
because self-antigens
are always present but
foreign antigens are
transient
Before clonal deletion of
an anti-self B cell, the
cell can attempt receptor
editing of the light chain
Fig 7.26
RAGs expression
is still on
So, light chain can use repeated
rearrangements and can receptor
edit. Repeated rearrangements are
to make a functional molecule
whereas receptor editing is to
avoid clonal deletion of anti-self
specific B cells.
Immature B cell “edits” light
chain if it binds antigen (gets
negative selection signal). This
could rescue the cell from
negative selection (i.e., death).
Immature T cells continues to
rearrange a chain until the cell gets
positive selection signal. (will
eventually die if it does not receive
positive selection in a few days).
(needs signal to edit)
(needs signal to stop “editing”)
Figure 7-18
Allelic exclusion
Figure 3-11
Figure 4-12
Va are mixed with Vd
Figure 4-15
In a TCR a locus there are about 70 V gene segments and 60 J segments.
This provides for many attempts at a productive rearrangement. Rearrangements
stop when there is positive selection.
Figure 7-12
Figure 4-2
Mechanisms contributing to generation of primary antibody
diversity in humans
VxJx(D)
H-L chain associations
200
Hxk
Hxl
120
6000
1,200,000
720,000
Levels of Regulation of Ig Gene
Expression & GOD
1.
2.
3.
4.
V-(D)-J rearrangement
Class switch recombination (CSR)
Somatic hypermutation (SH)
Receptor editing\revision
V(D)J Recombination : RAG
RAG 1 & 2 required
RAG 1 or RAG 2 K.O. mice
Also SCID in Man
No VDJ recombination (B or T-cells)
No dsDNA breaks
RAG function
• RAG 1 & 2 transfected into fibroblasts +
synthetic V-D-J substrate
Recombines exons
Transcription control
In synthetic substrate transfected into cell lines
and mice, deletion of any of promoters or
enhancers blocks rearrangement
Transcription Controls V(D)J
Recombination
P
Pi
Lp
D
v
Pi
J
Ei
3’Eμ
Cμ
Gene construct to test control elements: Deletion of any
of Promoters (P or Pi) or Enhancers (Ei or 3’Eμ blocks
recombination.
Nucleotide sequences of TCR V-gamma6 / J-gamma1
junction
Vg6:
TGG GAT A cactcta…………
…………cactgtg AT AGC
Jg1:
P
VJ1
VJ2
VJ3
VJ4
VJ5
VJ6
VJ7
N
P
V
J
TGG GAT
TGG GA
TGG GA
TGG GAT
TGG GA
TGG GAT A
TGG GAT
T AGC
AGC
AT AGC
GC
AT AGC
T AGC
AT AGC
CCG
TGG
AT
T
TT
AT
Generation of diversity
Mechanism of
action of RAG1
& RAG2
N addition by TdT=Terminal
deoxynucleotidyl transferase
RSS
RSS
5’ N-O-P-O-N 3’
3’ N-O-P-O-N 5’
Antibody protein sequence is altered in
several independent ways
•Regulation of membrane vs secreted IgM RNA splicing
•(Pre-immune) membrane IgM/IgD co-expression RNA splicing
AID dependent (activation induced cytidine deaminase):
•Affinity maturation
Somatic hyper-mutation
•Isotype switch: Switch to IgG, IgA, IgE antibody H-chain C exons
•Gene conversion
Note: there are exons encoding the membrane and secreted
forms of each of the antibody heavy chains.
Prior to activation, B cells express
two forms of antibody as
membrane receptors, IgM and
IgD. On any given cell, the
antigen specificity is identical.
This is accomplished by
differential RNA splicing.
IgM
IgD
Antibody protein sequence and bioactivity changes during
the immune response
Low affinity
IgM
High affinity
IgG
IgM
Somatic hypermutation introduces diversity BCR (not in TCR)
Figure 4-9
Mutations
silent Phe ttc
ttt
neutral
Lys aaa
Arg aga
Positive
Glu gaa
Gly gga
deleterious
Ser tca
Stop taa
Affinity maturation
Selection of clones with
the highest affinity
Characteristics of Somatic Mutation
only in B cells and not in T cells
1. Occurs at high rates: 10 -4 -10 -3 /bp/generation.
2. Occurs by untemplated single base substitutions.
3. Restricted to a brief period of B cell differentiation.
4. Restricted to the rearranged V region and its immediate
flanking sequences.
5. Occurs in germinal centers with T cell help.
6. Occurs throughout the V region but more frequently in
RGYW (A/G G C/T A/T) motifs.
BioEssays 20:227–234, 1998
The mutation Domain
• Mutations largely confined to variable regions
• Occur over a 1-2 kB region around the rearranged VJ
gene segment
• Found predominantly in CDRs but due to selection
Hyper-mutations spread in the V(D)J region
Patricia J. Gearhart and Richard D. Wood
Figure 3-6
CDR1 CDR2
CDR3
CDR1
CDR2
CDR3
Requirements for recruiting SHM
(Somatic Hyper Mutation)
• In kappa locus both intronic and 3’ enhancer
elements are required
• Transcription is required, mutations decrease with
distance from the promoter
• Expression of AID (activation-induced cytidine
deaminase) is essential
Activation Induced cytidine Deaminase (AID)
induces somatic hypermutation
A
T
C
G
AID
Base excision repair of G-T mismatch
removes base leaves sugar backbone
Mismatch excision repair of newly replicated DNA
Ig Class Switch
Recombination (CSR)
Figure 4-21
.
CSR occurs by an intrachromosomal deletional recombination between
switch (S) region sequences located upstream of the constant region
genes. S region sequences consist of tandem repeats of short (20–80 bp)
consensus elements, extending from 2 to 10 kb in length, and
recombination can occur at any site within the S regions. The process is
thought to be initiated by the creation of double-strand breaks within the S
regions, consistent with the ability to detect the deleted DNA as a circle.
Although the different S regions have short sequence elements in common
(e.g., GGGGT, GGGCT, or GAGCT), they differ too much to undergo
homologous recombination and CSR is thought to occur by a type of
nonhomologous end joining (NHEJ).
Class Switch Recombination
• Transcription in switch region required
• Cytokine directs transcription to I exon
promoter
• RNA/DNA hybrid of product flags target
site for recombination
Ig Class Switch Recombination (CSR)
Initiated by CD40L:CD40 + appropriate cytokine
Cytokine
IgG1
IL-4
+
IgG2a IgG2b IgG3
TGFa
IgE
+
IL-5
IFNg
IgA
+
+
+
+
+
VDJ joining creates heavy chain variable region domain
a
VD
Figure 9-8
Ig Class Switch Recombination
Evidence:
• CSR blocked by deletion of :– Splice site
– I exon promoter
– I exon
•
RNA/DNA hybrids identified
Ig Class Switch Recombination
• RAG 1 & 2 NOT required
• CSR requires:– Activation-induced cytidine deaminase (AID,
mice & hu.)
– MSH2 (K.O. mice)
Ig Class Switch Recombination
• AID initiates staggered dsDNA breaks
• Cut by endonuclease
• Breaks are repaired by error-prone DNA
pol.
Class Switch Recombination
• DNA breaks at switch
sites are staggered
• Breaks are repaired by
error-prone DNA pol. &
ligation
Class Switch Recombination
Evidence:• AID- - hu. & AID or MSH2 K.O. mice  No
CSR
Birds
Rabbits
Cows
Pigs
Sheep (SHM)
horses
Figure 4-13
AID converts C to U
Somatic Hypermutation (SHM) & Class
Switch Recombination (CSR)
Activation of B lymphocytes by antigen and costimulatory signals,
usually from T lymphocytes, initiates two processes of antibody
diversification. Somatic hypermutation (SHM) introduces mutations
in the variable region genes, which, in conjunction with antigen
selection, generates antibodies with increased affinity.
The second process, class switch recombination (CSR), enables B
Cells to diversify the constant region and thereby the effector
function of the antibody molecule, while maintaining the same
antigen-binding domain.
These two mechanisms have several shared features but both are
poorly understood
Activation-induced cytidine deaminase (AID) is essential for both
SHM and CSR
The data indicate its role is to convert dC to dU residues within variable
genes and S regions. Resolution of the dU residues could introduce
mutations that are characteristic both of SHM and of segments
surrounding S–S junctions. Furthermore, removal of the dU residues by
the base excision repair (BER) pathway could introduce the DNA breaks
necessary to initiate CSR. This has not been demonstrated; however,
CSR is 90% reduced in B cells from mice deficient in the BER enzyme
uracil DNA glycosylase (UNG) that excises dU residues, and even more
severely reduced in some patients with hyper IgM syndrome caused by
deleterious mutations in UNG.
Class Switch Recombination
Cytokines direct transcription of I-S-CH which initiates CSR