1. SOMATIC HYPERMUTATION

2. CDR1 CDR2 CDR3 VL J2 gene product V35 gene product
Complementary Determining Region = hypervariable region

3. STRUCTURE OF THE VARIABLE REGION
Hypervariable (HVR) or complimentarity determining regions (CDR)
HVR3
FR1
FR2
FR3
FR4
HVR1
HVR2
Variability Index 25 75 50
100
Amino acid residue
150
Framework regions (FR)

5. Szomatikus hipermutáció
Variabilitás
FR1
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?

6. LIGHT CHAIN Light chain Disulphide bridges Heavy chain VL CL
FR FR FR FR4
CDR1 CDR CDR3
Heavy chain
VL CL
FR FR FR FR4
CDR1 CDR CDR3

7. SOMATIC HYPERMUTATION Hypervariable regions
Day 0. Ag
Plasma cell clones 1 2 3 4 5 6 7 8
Day 7
PRIMARY
immune response
AFFINITY MATURATION 9
1011 12 13 14 15 16
Day 14
Day 14. Ag 17
1819 20 21 22 23 24
Day 21
SECONDARY
Immune response
Hypervariable regions

8. Hypermutation occurs under the influence of activated T cells
Somatic hypermutation leads to affinity maturation
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
CDR1
CDR2
CDR3
Day 6
CDR1
CDR2
CDR3
Day 8
Day 12
Day 18
Deleterious mutation
Beneficial mutation
Neutral mutation
Lower affinity - Not clonally selected
Higher affinity - Clonally selected
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.

9. 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

10. 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 10

11. Where and how do all these things take place?
B – CELL ACTIVATION
Where and how do all these things take place?

13. B-cell recycling in the absence of antigen
(lymph node)
B cells
in blood
T cell area
B cell area
Efferens
lymph

14. Recirculating B cells are trapped by foreign antigens in lymphoid organs
B cells leave blood & enter lymph node via
high endothelial venules
B cells
proliferate
rapidly
Antigen enters
node in afferent
lymphatic Y
Germinal centre
releases B cells
that differentiate
into plasma cells
GERMINAL CENTRE
Transient structure of
Intense proliferation

15. Germinal Center Reaction
Germinal centers, where B cells proliferate and undergo both isotype switching and somatic hypermutation, form within the B cell follicles in lymphoid organs. The formation of germinal centers starts when dendritic cells displaying antigen on their surface activate antigen-specific CD4 T cells, which proliferate and mature into effector cells capable of activating antigen-specific B cells. Once activated by a CD4 T cell, the B cell proliferates to form a primary focus of antigen specific B cells. B cells from the primary focus migrate to nearby follicles and proliferate. Other B cells from the primary focus persist in the T cell area for a short while, secreting antibody, but eventually die. The B cells that enter the follicle begin to proliferate rapidly. During this time they also undergo somatic mutation, to introduce new variation into the B cell receptor. B cells undergo a process of selection after somatic mutation, when the receptors are tested for their ability to bind antigen. Those that fail to bind, or that fail to compete efficiently against other B cell receptors, die.

16. „Dating” in the peripheral lymphoid organs

17. The structure of the germinal centre
Somatic hypermutation LZ
FDC DZ
Somatic hypermutation
LZ: light zone
DZ: dark zone
FDC: follicular dendritic cell

18. Antigen is bound on the surface of follicular dendritic cells (FDC)
 FDC-s bind immune complexes (Ag-Ab )
 Ag detectable for 12 months following immunization
 A single cell binds various antigens
Fig 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
B cells recognize Ag on the surface of FDC
This concept is being challenged now, and there has been some backtracking in the current text. It is possible that the FDCs provide persistent Ag to long-lived plasma cells.

19. T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS
IgM
IgG
IgA
IgE

20. SIGNALING UNITS OF THE B-CELL RECEPTOR
Ig-a/CD79a a b Y
Ig-b/CD79b
Ig domain + CHO
ITAM
ITAM: YxxL x7 YxxI
ITAM: Immunoreceptor Tyrosine-based Activation Motif

21. Main steps of B-cell signal transduction

22. CONSEQUENCES OF B-CELL RECEPTOR CROSS LINKING
Ag binding, cross-linking of surface Ig
Lymphocyte activation
Phenotypic/
Functional change
Fokozott túlélés
Osztódás
Ko-stimuláló molekulák kifejeződése
Citokin receptorok kifejeződése
Kivándorlás a limfoid tüszőből a T sejtes területekre

23. KINETICS OF LYMPHOCYTE ACTIVATION
ANTIGEN
SIGNAL1.
Nyugvó limfocita G0
sejtosztódás
DNA synthesis
Effector cell Memory cell
Transport
Membrane change
RNA and protein synthesis
Resting lymphocyte G0
Ko-receptor
Adhesion molecule
Cytokines
SIGNAL2.
Resting lymphocyte G0
PTK activation RNA synthesis
Free Ca Protein synthesis
Protein phosphorylation DNA synthesis
Lymphoblast
0 10sec 1min 5min hr hrs hrs hrs

24. Antigenic
determinant
C3d
THE CO-STIMULATORY ROLE OF CR2 (CD21) COMPLEMENT RECEPTOR IN B – LYMPHOCYTES
ANTIGÉN
CD21/CR2
CD19 Y
TAPA=CD81
B-CELL
Enhanced B-cell activation

25. THE NEURAMIC ACID RECEPTOR CD22 INHIBITS ACTIVATION THROUGH THE A B-CELL RECEPTOR
Tissue cells
Bacterium
Mannose
Neuraminic acid
B Cell
Antigen
CD22
Inhibited B cell activation

26. 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
PLASMA CELL
OPSONIZATION
Binding of antibody increases phagocytosis
FcR
FcR CR1
Complement
C3b
COMPLEMENT ACTIVATION
Opsonization by C3b
PHAGOCYTES
ENGULFMENT, DEGRADATION

27. 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

28. Growth factors
MHC – peptid - TCR
Adhesion molecules
ANTIBODIES
Affinity