SOMATIC HYPERMUTATION

CDR1CDR2CDR3 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 HVR1 HVR2 Variability Index Amino acid residue Framework regions (FR)

Szomatikus hipermutáció FR1FR2FR3FR4CDR2CDR3CDR1 Aminsav szám Variabilitás 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?

CDR1 CDR2 CDR3 Light chain Heavy chain CDR1 CDR2 CDR3 VLCL LIGHT CHAIN Disulphide bridges FR1 FR2 FR3 FR4

SOMATIC HYPERMUTATION Day 0. Ag Day 14. Ag PRIMARY immune response SECONDARY Immune response AFFINITY MATURATION Day 21 Day 14 Day 7 Hypervariable regions Plasma cell clones

Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 CDR1CDR2CDR3 Day 6 CDR1CDR2CDR3CDR1CDR2CDR3CDR1CDR2CDR3 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 Somatic hypermutation leads to affinity maturation 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.

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

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 Efferent lymph T cell area B cell area

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

Germinal Center Reaction

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

„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 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 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-  /CD79a Ig-  /CD79b ITAM: Immunoreceptor Tyrosine-based Activation Motif   Y Y Y Y ITAM ITAM Ig domain + CHO SIGNALING UNITS OF THE B-CELL RECEPTOR ITAM: YxxL x7 YxxI

Main steps of B-cell signal transduction

CONSEQUENCES OF B-CELL RECEPTOR CROSS LINKING Ag binding, cross-linking of surface Ig Lymphocyte activation Phenotypic/ Functional change

KINETICS OF LYMPHOCYTE ACTIVATION ANTIGEN SIGNAL1. Ko-receptor Adhesion molecule Cytokines SIGNAL2. Resting lymphocyte G 0 PTK 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 G 0 sejtosztódás DNA synthesis Effector cell Memory cell Transport Membrane change RNA and protein synthesis Resting lymphocyte G 0

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

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

EFFECTOR FUNCTIONS OF ANTIBODIES PLASMA CELL NEUTRALIZATION Small proportion of antibodies INHIBITION Binding of bacteria to epithelial cells Binding of viruses to receptor Binding of bacterial toxins to target cells OPSONIZATION Binding of antibody increases phagocytosis Fc  R Fc  R CR1 Complement C3b COMPLEMENT ACTIVATION Opsonization by C3b PHAGOCYTES ENGULFMENT, DEGRADATION

SIZE SHAPE HYDROPHOBIC HYDROPHYLIC POSITIVELY CHARGED NEGATIVELY 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

Affinity ANTIBODIES Growth factors MHC – peptid - TCR Adhesion molecules