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B-CELL DIFFERENTIATION IN THE PERIPHERY
Ag
Ag
Ag
Memory B-cell
Activated B-cell
Mature naive B-cell
ISOTYPE
SWITCH
SOMATIC
HYPERMUTATION
BONE
MARROW
3
2
1
4
n
5
Potential B-cell
repertoire
Self recognition
Clonal deletion
Self structure
PERIPHERAL
LYMPHOID
ORGANS
3
2
Available B-cell
repertoire
n
4
Antigen dependent
Clonal division
Antigen – non-self
3
3
3
3
3
3
3
3
3
3
3
3
3
Memory cell
repertoire
3
3
3
3
3
3
3
3
3
Effector cell
repertoire
The molecular genetics of immunoglobulins
•
If the BCR and the soluble antibodies are identical, by what
mechanism switch from one to the other is controlled?
MEMBRANE VS SECRETED IMMUNOGLOBULIN
•
By what mechanism are antibodies with the same specificity but with
different isotypes generated?
ISOTYPE SWITCH
•
How could antibodies increase their affinity in the course of the
immune response?
SOMATIC HYPERMUTATION
MEMBRANE BOUND AND SECRETED
IMMUNOGLOBULIN
The constant region has additional optional exons
Cm
Primary transcript RNA
Each domain of the H
chain is encoded by a
separate exon
Cm1
Cm2
AAAAA
Secretion
coding
sequence
Cm3
Polyadenylation
site (secreted)
pAs
Polyadenylation
site (membrane)
pAm
Cm4
Membrane
coding
sequence
Membrane IgM constant region
DNA
Cm1
Cm2
Cm3
Cm4
Transcription
1° transcript
pAm
Cm1
Cleavage &
polyadenylation at pAm
and RNA splicing
mRNA
Cm2
Cm3
Cm1 Cm2 Cm3 Cm4
Cm4
AAAAA
AAAAA
Membrane coding
sequence encodes
transmembrane region
that retains IgM in the
cell membrane
Protein
Fc
Secreted IgM constant region
DNA
Cm1
Cm2
Cm3
Cm4
Transcription
1° transcript
pAs
Cm1
Cm2
Cm3
Cm4
AAAAA
Cleavage polyadenylation
at pAs and RNA splicing
mRNA
Cm1 Cm2 Cm3 Cm4
Protein
AAAAA
Secretion coding
sequence encodes the
C terminus of soluble,
secreted IgM
Fc
ISOTYPE SWITCH
Antibody isotype switching
Throughout the immune response the specificity of an antibody will
be essentially the same (notwithstanding affinity maturation)
The effector function of antibodies throughout a response needs to
change drastically as the response progresses.
Antibodies are able to retain Variable regions whilst exchanging
Constant regions that contain the structures that interact with cells.
Organisation of the functional human heavy chain C region genes
J regions
Cm
Cd
Cg3
Cg1 Ca1 Cg2
Cg4
Ce
Ca2
embrionális
Embryonal
L1 V1 L2 V2 Ln Vn D1 - 12
DNS
DNA
5'
szomatikus
rekombináció
Somatic
recombination
D–
D-J kapcsolódás
átrendeződött
Rearranged
DNA
DNS
L1
V1
Ln
Vn
Cm Cδ Cg3
CM CD CG3
J1-4
J
CA1Cε2
Ca1
CG1
CE2Cg1
CE1 Ca2
CG
2 CG
4 Cε1
CA2
C
g1
Cg4
D1D2 J1J2-4 Cm
CM Cδ
CD
5'
3'
szomatikus rekombináció
Somatic
recombination
V
-D-J kapcsolódás
CM Cδ
CD
D2J1 J2-4 Cm
3'
L1 V1
5'
Primerprimer
RNA transcript
RNS-átirat
mRNA
mRNS
3’
Transcription
transzkripció
5'
L1 V1D2 J1 J2-4 Cm
CM
Cδ
CD
IgM
Cγ1
IgG
Cγ2
IgG
Cγ3
IgG
Modification Cγ4
IgG
AAAA
transzláció
Translation
L V DJ Cm
C
polipeptid
Ig ISOTYPES
Cµ
3'
Processing
átalakítás
Cm
L1 V1 D2 J1 CM
naszcens
Nascent
polypeptide
módosítás
V
C
V–D–J
NEHÉZL
ÁNC (M
)
Heavy
chain
IgM
Cα
IgA
Cε
IgE
Switch regions
Cm
Sm
Cd
Cg3
Sg3
Cg1
Sg1
Ca1
Sa1
Cg2
Sg2
Cg4
Sg4
Ce
Se
Ca2
Sa2
• Upstream of C regions are repetitive regions of DNA called switch regions.
(The exception is the Cd region that has no switch region).
• The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is
between 3 and 7.
• Switching is mechanistically similar in many ways to V(D)J
recombination, but
• All recombination events are productive
• Different recombination signal sequences and enzymes are involved
• Requires antigen stimulation of B cell
• Not a random event, but regulated by external signals such as T cell
derived cytokines
• Isotype switching does not take place in the bone marrow, but occurs after
B cell activation in the peripheral lymphoid organs
Switch recombination
Cm
Cd
Cg3
Cg1
Ca1
Cg2
Cg4
Cd
Ce
Cd
Ca2
Sg3
Cg3
Cg3
Cm
Sg1
Cm
Cg1
V23D5J4
Cg3
V23D5J4
Ca1
V23D5J4
Ca1
V23D5J4
Cg3
V23D5J4
Ca1
V23D5J4
Ca1
IgG3 produced.
Switch from IgM
IgA1 produced.
Switch from IgG3
IgA1 produced.
Switch from IgM
At each recombination constant regions are deleted from the genome
An IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1
Model for Class Switch Recombination (CSR)
AID (Activation Induced (citidin) Deaminase C →U, RNA editing enzyme)
UNG excises U → abasic sites, AP-endonuclease/lyase activity → ss nicks
Class switch defects - Hiper IgM syndrome type 2 in humans (autosomal)
•HYPER IgM SYNDROME (Autosomal)
-Intrinsic B cell defect, activation induced deaiminase
(AID) deficiency. Cytidine uridine conversion.
-The enyme is involved in affinity maturation and
Ig. class switch
Lack of germinal centers in lymph nodes of
X-linked Hyper-IgM syndrome patients
SOMATIC HYPERMUTATION
VL
J2 gene product
V35 gene product
CDR1
CDR2
CDR3
Complementary Determining Region = hypervariable region
STRUCTURE OF THE VARIABLE
REGION
• Hypervariable (HVR) or complimentarity
determining regions (CDR)
HVR
3
Variability
Index
15
0
10
0
5
0
0
HVR
HVR1 2
FR1
25
FR
2
FR
3
7
50
Amino acid 5
residue(FR)
• Framework regions
FR
4
10
0
100
Light
chain
90
80
70
60
50
40
LIGHT CHAIN
30
20
10
NH2
0
10
FR1
20
30
40
FR2
CDR1
50
60
70
FR3
80
CDR2
90 100 110 120
FR4
Disulphide
bridges
CDR3
COOH
100
90
Heavy
chain
80
70
60
50
VL
40
30
20
10
0
10
20
FR1
30
40
50
FR2
CDR1
60
70
80
FR3
CDR2
90 100 110 120
FR4
CDR3
CL
Hypervariable loops and framework: Summary
• The framework supports the hypervariable loops
• The framework forms a compact b barrel/sandwich with a
hydrophobic core
• The hypervariable loops join, and are more flexible than,
the b
strands
• The sequences of the hypervariable loops are highly
variable amongst antibodies of different specificities
• The variable sequences of the hypervariable loops
influences
the shape, hydrophobicity and charge at the
tip of the antibody
• Variable amino acid sequence in the hypervariable loops
accounts for the diversity of antigens that can be
recognised by a repertoire of antibodies
SOMATIC HYPERMUTATION
Day 0.
Ag
Day7 7nap
CDR1 CDR2 CDR3
CDR1 CDR2
IgM
PRIMARY
immune
response
Day
14 14
nap
IgM/IgG
Day 14.
Ag
Day
21 21
nap
CDR3
1
2
3
4
5
6
7
8
AFFINITY
9
MATURATI
10
11
ON
12
13
14
15
16
IgG
SECONDARY
Immune
Plasma cell
clones
Hypervariable regions
17
18
19
20
21
22
23
24
Somatic hypermutation leads to affinity maturation
Day 6
Day 8
Day 12
Day 18
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
CDR1
CDR2
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
Deleterious mutationLower affinity - Not clonally selected
Beneficial mutation Higher affinity - Clonally selected
Neutral mutation
Identical affinity - No influence on clonal selection
Hypermutation occurs under the influence of activated T cells
Mutations are focussed on ‘hot spots’ (i.e. the CDRs) and are due to double
stranded breaks repaired by an error prone DNA repair enzyme.
FR1
CDR1 FR2 CDR2
FR3
CDR3
FR4
H-CHAIN
CDR2
CDR1
100
CDR3
Variability
80
60
40
Antigén determináns
20
CDR1
CDR2
CDR3
L-CHAIN
20
40
60
80
100
120
Amino acid No.
Wu - Kabat analysis compared point
mutations in Ig of different specificity.
CDR1 and CDR2 regions are encoded by the V-gene
The CDR3 of L-chain is encoded by V and J
SOME CHARACTERISTICS OF SOMATIC HYPERMUTATIONS
Mutations made by AID (same enzyme as for
class switching)
Both CSR and SHM requires strand brakes
10-3 / Bp mutation rate (a million times more
than expected)
Both CSR and SHM occurs in germinal center Bcells
Very much site specific, CDR regions of the BCR
(some other genes too, but limited (CD95)