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SOMATIC HYPERMUTATION. VL. J2 gene product. V35 gene product. CDR1. CDR2. CDR3. C omplementary D etermining R egion = hypervariable region. STRUCTURE OF THE VARIABLE REGION. H y pervariable (HVR) or complimentarity determining regions (CDR). HVR3. 150. Variability Index. 100. HVR2. - PowerPoint PPT Presentation
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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)
Szomatikus hipermutáció
FR1 FR2 FR3 FR4CDR2 CDR3CDR1
Aminsav szám
Variabilitás
80
100
60
40
20
20 40 60 80 100 120
A különböző specificitású ellenanyagokban található pont mutációk összehasonlítása
Wu - Kabat analízissel
Mik a következményei az immunválasz során végbemenő mutációknakegy adott epitóp ellen irányuló ellenanyagban?
Hogyan befolyásolja az ellenanyag specificitását és affinitását?
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
B – CELL ACTIVATION
Where and how do all these things take place?
B-cell recycling in the absence of antigen (lymph node)
B cells in blood
Efferentlymph
T cell area
B cell area
Antigen entersnode in afferent
lymphatic
Y
Y
Y
Y
Y
YY
Y
Y
Y
Y
Y
Y
Y
YY
Y
YB cells leave blood & enter lymph node via
high endothelial venulesB cellsproliferate
rapidly
GERMINAL CENTRETransient structure ofIntense proliferation
Germinal centrereleases B cellsthat differentiateinto plasma cells
Recirculating B cells are trapped by foreign antigens in lymphoid organs
Germinal Center Reaction
T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANSORGANS
IgM
IgGIgAIgE
„Dating” in the peripheral lymphoid organs
Antigen-stimulated B cells become trapped in the T-cell zone
The primary foci and secondary follicle formation
The structure of the germinal centre
Somatic hypermutation
FDC
Somatic hypermutation
DZ
LZ
LZ: light zoneDZ: dark zoneFDC: 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
Ig-Ig-/CD79a/CD79a Ig-Ig-/CD79b/CD79b
ITAM: ITAM: IImmunoreceptor mmunoreceptor TTyrosineyrosine--based based AActivation ctivation MMotifotif
Y
Y
Y
YITAMITAM ITAMITAM
Ig domain + CHOIg domain + CHO
SIGNALING UNITS OF THE B-CELL RECEPTOR
ITAM:ITAM: Y YxxxxLL x7x7 YYxxxxII
Main steps of B-cell signal transductionMain steps of B-cell signal transduction
CONSEQUENCES OF B-CELL RECEPTOR CROSS LINKINGCONSEQUENCES OF B-CELL RECEPTOR CROSS LINKING
Ag binding, cross-linking of
surface IgLymphocyte
activationPhenotypic/
Functional change
KINETICS OF LYMPHOCYTE ACTIVATION
ANTIGEN SIGNAL1.
Ko-receptorAdhesion molecule
Cytokines SIGNAL2.
Resting lymphocyte G0PTK activation RNA synthesis Free Ca++ Protein synthesis Protein phosphorylation DNA synthesis
Lymphoblast
0 10sec 1min 5min 1hr 6 hrs 12 hrs 24 hrs
Nyugvó limfocita G0
G1
G2
M
Ssejtosztódás
DNA synthesis
Effector cell Memory cell
Transport Membrane changeRNA and protein synthesis
Resting lymphocyte G0
AntigAntigenicenicdeterminantdeterminant
C3C3dd
THE THE CO-STIMULATORYCO-STIMULATORY ROLE OF ROLE OF CR2 (CD21) CR2 (CD21) COMPLEMENT RECEPTOR IN B – LYMPHOCYTESCOMPLEMENT RECEPTOR IN B – LYMPHOCYTES
ANTIGÉN
CD21CD21/CR2/CR2
CD19CD19
YY
TAPA=CD81TAPA=CD81
Enhanced B-cell activation
BB-CELL-CELL
THE NEURAMINIC ACID RECEPTOR CD22 INHIBITS THE NEURAMINIC ACID RECEPTOR CD22 INHIBITS ACTIVATION THROUGH THE A B-CELL RECEPTOR ACTIVATION THROUGH THE A B-CELL RECEPTOR
B B CellCellAntigAntigeenn
Tissue cells
BaBacctteeriumrium
MannMannoseose
CD22CD22
Neuraminic acid
Inhibited B cell activation
EFFECTOR FUNCTIONS OF ANTIBODIES
PLASMA CELL
NEUTRALIZATION
Small proportion of antibodies
INHIBITIONBinding of bacteria to
epithelial cellsBinding of viruses to
receptorBinding of bacterial toxins to target cells
OPSONIZATION
Binding of antibody increases phagocytosis
FcR
FcR
FcR CR1
ComplementC3b
COMPLEMENT ACTIVATION
Opsonization by C3b
PHAGOCYTES
ENGULFMENT, DEGRADATION
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