Transcript 1. dia - immunology.unideb.hu

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)
Variability Index
HVR3
150
100
HVR2
HVR1
50
FR2
FR1
0
25
FR3
75
50
Amino acid residue
• Framework regions (FR)
FR4
100
Szomatikus hipermutáció
FR1
Variabilitás
CDR1 FR2 CDR2
FR3
CDR3
FR4
100
80
60
40
20
20
40
60
80
100
120
Aminsav szám
A különböző specificitású ellenanyagokban
található pont mutációk összehasonlítása
Wu - Kabat analízissel
Mik a következményei az immunválasz során végbemenő mutációknak
egy adott epitóp ellen irányuló ellenanyagban?
Hogyan befolyásolja az ellenanyag specificitását és affinitását?
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
Disulphide bridges
90 100 110 120
FR4
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
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
IgG
SECONDARY
Immune response
Hypervariable regions
Plasma cell
clones
CDR3
1
2
3
4
5
6
7
8
AFFINITY
MATURATION
9
10
11
12
13
14
15
16
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 mutation Lower 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
The CDR3 of H-cain is encoded by V, D and J genes
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
B – CELL ACTIVATION
Where and how do all these things
take place?
B-cell recycling in the absence of antigen
(lymph node)
T cell area
B cells
in blood
B cell
area
Efferens
lymph
Recirculating B cells are trapped by foreign
antigens in lymphoid organs
Antigen enters
node in afferent
lymphatic
YY
Y
B cells
proliferate
rapidly
B cells leave blood &
enter lymph node via
high endothelial venules
Y
YY
Y
Y
Y
GERMINAL CENTRE
Transient structure of
Intense proliferation
YY
Y
Germinal centre
releases B cells
that differentiate
into plasma cells
Germinal Center Reaction
„Dating” in the peripheral lymphoid organs
The structure of the germinal centre
Somatic hypermutation
LZ
FDC
DZ
Somatic hypermutation
LZ: light zone
DZ: dark zone
FDC: follicular dendritic cell
Antigen is bound on the surface of follicular dendritic cells (FDC)
FDC
 FDC-s bind immune complexes (Ag-Ab )
 Ag detectable for 12 months following immunization
 A single cell binds various antigens
B cells recognize Ag on the surface of FDC
Fig. 9.15. On the surface of FDC-s immune
complexes form the so-called iccosomes,that can be
released and taken up later by the surrounding
germinal center B cells
T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID
ORGANS
IgM
IgG
IgA
IgE
SIGNALING UNITS OF THE B-CELL RECEPTOR
Ig-a/CD79a
Ig-b/CD79b
a b
Y
ITAM
Y
Ig domain + CHO
Y
Y
ITAM
ITAM: YxxL x7 YxxI
ITAM: Immunoreceptor Tyrosine-based Activation Motif
Main steps of B-cell signal transduction
CONSEQUENCES OF B-CELL RECEPTOR CROSS LINKING
Ag binding,
Phenotypic/
cross-linking of Lymphocyte
activation Functional change
surface Ig
KINETICS OF LYMPHOCYTE ACTIVATION
ANTIGEN
SIGNAL1.
Resting limfocita
lymphocyte
Nyugvó
GG
0 0
Effector cell
Memory cell
G1
Ko-receptor
Adhesion molecule
Cytokines
SIGNAL2.
Transport
Membrane change
RNA and protein synthesis
M
sejtosztódás
G2
S
DNA synthesis
Lymphoblast
Resting lymphocyte G0
PTK activation
RNA synthesis
Free Ca++
Protein synthesis
Protein phosphorylation
DNA synthesis
0 10sec 1min 5min
1hr
6 hrs
12 hrs
24 hrs
THE CO-STIMULATORY ROLE OF CR2 (CD21)
COMPLEMENT RECEPTOR IN B – LYMPHOCYTES
C3d
ANTIGÉN
Antigenic
determinant
CD21/CR2
CD19
B-CELL
TAPA=CD81
Y
Y
Enhanced B-cell activation
THE NEURAMIC ACID RECEPTOR CD22 INHIBITS
ACTIVATION THROUGH THE A B-CELL RECEPTOR
Mannose
Tissue cells
Bacterium
Neuraminic acid
Antigen
B Cell
CD22
Inhibited B cell activation
EFFECTOR FUNCTIONS OF ANTIBODIES
INHIBITION
Binding of bacteria to
epithelial cells
Binding of viruses to
receptor
Binding of bacterial
toxins to target cells
NEUTRALIZATION
Small proportion of
antibodies
OPSONIZATION
COMPLEMENT
ACTIVATION
Binding of antibody
increases phagocytosis
PLASMA CELL
FcR
Opsonization by C3b
Complement
C3b
FcR
FcR CR1
PHAGOCYTES
ENGULFMENT, DEGRADATION
FEATURES OF THE
BINDING SITE
SIZE
SHAPE
HYDROPHOBIC
HYDROPHYLIC
POSITIVELY CHARGED
NEGATIVELY CHARGED
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
KD=
[A] [B]
[AB]
10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 M
Growth factors
MHC – peptid - TCR
CD2/LFA-3 Adhesion molecules
CD28/B7
LFA-1/ICAM-1
ANTIBODIES
Affinity