a a activated b-cell mature naive b-cell memory b-cell b-cell differentiation in the periphery...
TRANSCRIPT
Activated B-cellMature naive B-cell
Memory B-cell
B-CELL DIFFERENTIATION IN THE PERIPHERY
SOMATIC HYPERMUTATION
ISOTYPE SWITCH
Ag
AgAg
n42 3
PERIPHERAL LYMPHOID ORGANS
Available B-cell repertoire
n3 42 51BONE
MARROW
Potential B-cell repertoire
Self structure
Self recognitionClonal deletion
Antigen – non-self
Antigen dependentClonal division
Effector cell repertoire
Memory cell repertoire
• 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
The molecular genetics of immunoglobulins
MEMBRANE BOUND AND SECRETED IMMUNOGLOBULIN
Primary transcript RNA AAAAA
C
Polyadenylation site (secreted)
pAs
Polyadenylation site (membrane)
pAm
The constant region has additional optional exons
C1 C2 C3 C4
Each domain of the H chain is encoded by a
separate exonSecretion
codingsequence
Membranecoding
sequence
mRNAC1 C2 C3 C4 AAAAA
Transcription
Membrane IgM constant region
C1 C2 C3 C41° transcriptpAm
AAAAA
C1 C2 C3 C4DNA
Membrane coding sequence encodes
transmembrane regionthat retains IgM in the
cell membrane
Fc
Protein
Cleavage & polyadenylation at pAm and RNA splicing
mRNA
Secreted IgM constant region
C1 C2 C3 C4 AAAAA
C1 C2 C3 C4DNA
Cleavage polyadenylation at pAs and RNA splicing
1° transcriptpAs
C1 C2 C3 C4
Transcription
AAAAA
Secretion coding sequence encodes the C terminus of soluble,
secreted IgM
Fc
Protein
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.
J regions C2CC4C2C1C1C3CC
Organisation of the functional human heavy chain C region genes
átrendeződöttDNS
primerRNS-átirat
mRNS
naszcenspolipeptid
átalakítás
transzláció
módosítás
5'
AAAA
3'L1 V1D2J1J2-4 CM CD
transzkripció
V C
L V DJ C
L1 V1D2J1 CM
5' 3'
szomatikus rekombinációV-D-J kapcsolódás
5' 3'
L1 V1 Ln Vn D1D2J1J2-4 CM CD
L1 V1 D2J1 J2-4 CM CD
NEHÉZLÁNC (M)
szomatikus rekombinációD-J kapcsolódás
embrionálisDNS
L1 V1 Ln Vn J1-4 CM CDD1 - 125'
3’
L2 V2 C 3G
CE2 C 1G
CG2
CA1
CG4 CE1 CA2
C Cδ C3
C1 Cε2 C1 C 1 C4 Cε1 C2
C Cδ
C Cδ
C Cδ
IgM
C
C
Embryonal DNA
Rearranged DNA
Primer RNA transcript
mRNA
Nascent polypeptide
Somatic recombination D – J
Somatic recombination V – D – J
Transcription
Processing
Translation
Modification
Ig ISOTYPES
Cµ IgM
Cγ1 IgG
Cγ2 IgG
Cγ3 IgG
Cγ4 IgG
Cα IgA
Cε IgEHeavy chain
C2CC4C2C1C1C3CC
Switch regions
• 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
S3 S1 S1 S2 S4 S S2S
• Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the C region that has no switch region).
• The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7.
C2CC4C2C1C1C3CC
C
C
C3V23D5J4
S3
C
C
C3
V23D5J4
C1
S1
C1
C3
V23D5J4 C1
C3V23D5J4
IgG3 produced.Switch from IgM
V23D5J4 C1
IgA1 produced.Switch from IgG3
V23D5J4 C1
IgA1 produced.Switch from IgM
Switch recombination
At each recombination constant regions are deleted from the genomeAn 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 opportunistic infections
SOMATIC HYPERMUTATION
CDR1CDR1 CDR2CDR2 CDR3CDR3
VL
Complementary Determining Region = hypervariable region
V35 gene product J2 gene product
STRUCTURE OF THE VARIABLE REGION
• Hypervariable (HVR) or complimentarity determining regions (CDR)
HVR3
FR1 FR2 FR3 FR4
HVR1HVR2
Var
iabi
lity
Ind
ex
25 7550 100Amino acid residue
150
100
50
0
• Framework regions (FR)
NH2
COOH
0 10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
110 120
0 10
10
20
20
30
30
40
40
50
50
60
60
70
70
80
80
90
90
100
100
110 120
CDR1 CDR2 CDR3
Light chain
Heavy chain
CDR1 CDR2 CDR3
VL CL
LIGHT CHAIN
Disulphide bridges
FR1 FR2 FR3 FR4
FR1 FR2 FR3 FR4
CDR1 CDR2 CDR3 CDR1 CDR2 CDR3
7 nap
14 nap
21 napIgG
IgM/IgG
IgM
SOMATIC HYPERMUTATIONDay 0.
Ag
Day 14. Ag
PRIMARY
immune response
SECONDARY
Immune response
AFFINITY MATURATION
Day 21
Day 14
Day 7
Hypervariable regions
Plasma cell clones
12345678
910111213141516
1718192021222324
Clone 1Clone 2Clone 3Clone 4Clone 5Clone 6Clone 7Clone 8Clone 9Clone 10
CD
R1
CD
R2
CD
R3
Day 6
CD
R1
CD
R2
CD
R3
CD
R1
CD
R2
CD
R3
CD
R1
CD
R2
CD
R3
Day 8 Day 12 Day 18
Deleterious mutationBeneficial mutationNeutral mutation
Lower affinity - Not clonally selectedHigher affinity - Clonally selectedIdentical affinity - No influence on clonal selection
Somatic hypermutation leads to affinity maturation
Hypermutation occurs under the influence of activated T cellsMutations are focussed on ‘hot spots’ (i.e. the CDRs) and are due to double stranded
breaks repaired by an error prone DNA repair enzyme.
CDR1 and CDR2 regions are encoded by the V-geneThe CDR3 of L-chain is encoded by V and J The CDR3 of H-cain is encoded by V, D and J genes
CDR1CDR2
CDR3
CDR1CDR2
CDR3
Antigén determináns
H-CHAIN
L-CHAINCDR1CDR2
CDR3
CDR1CDR2
CDR3
Antigén determináns
H-CHAIN
L-CHAIN
Antigén determinánsAntigén determináns
H-CHAIN
L-CHAIN
FR1 FR2 FR3 FR4CDR2 CDR3CDR1
Amino acid No.
Variability80
100
60
40
20
20 40 60 80 100 120
Wu - Kabat analysis compared point mutations in Ig of different specificity.
FR1 FR2 FR3 FR4CDR2 CDR3CDR1
Amino acid No.
Variability80
100
60
40
20
20 40 60 80 100 120
Amino acid No.
Variability80
100
60
40
20
20 40 60 80 100 120
Wu - Kabat analysis compared point mutations in Ig of different specificity.
•The framework supports the hypervariable loops
•The framework forms a compact barrel/sandwich with a
hydrophobic core
•The hypervariable loops join, and are more flexible than, the
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
Hypervariable loops and framework: Summary
SIZESHAPE
HYDROPHOBICHYDROPHYLIC
POSITIVELY CHARGEDNEGATIVELY CHARGED
FEATURES OF THE BINDING SITE
ANTIGEN BINDING IS MEDIATED BY
NON-COVALENT INTERACTIONS
One binding site is able to interact with more than
one antigen
The strength of interaction (affinity/avidity) varies in a
broad range
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 M
K =D [AB]
[A] [B]
C D2/LFA-3
C D28/B7 LFA-1/IC AM -1
AffinityANTIBODIES
Growth factors
MHC – peptid - TCR
Adhesion molecules