Where and how do all these things take place? 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 T cell area B cell area Efferens lymph

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

FDC depends on the presence of Antigen is bound on the surface of follicular dendritic cells (FDC) FDC  FDC bind immune complexes (Ag-Ab )  Ag detectable for 12 months following immunization  A single cell binds various antigens FDC depends on the presence of TNF-α, LTα, LTβ 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. On the surface of FDC immune complexes form the so-called iccosomes that can be released and taken up later by the surrounding germinal center B cells

„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

IMMUNOLOGICAL MEMORY – B CELLS Germinal Centre reaction proliferation somatic hypermutation affinity maturation Memory B cells previously activated passed affinity maturation present in the circulation rapidly proliferate and differentiate to plasma cell upon re-activation, or enter the GC reaction again Plasma cells provides serological memory: pre-existing neutralizing Abs to pathogens or toxins B B FDC B FDC B T B B B B B B plasma cell B B B B B T B

Somatic hypermutation Selection Repeated cycle Somatic hypermutation 9

T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANS BLIMP expresszió B-lymphocyte induced maturation protein IgM IgG IgA IgE

Regulation of memory vs plasma cell differentiation B cell T cell - activated Bcl-6 szuppresszor BLIMP gátló CD40 signaling 11

DEVELOPMENT OF B CELL MEMORY IN THE FOLLICLE Memory B cell apoptosis T CD40 CD40L Follicular dendritic cell (FDC) FcR CD21 Ag NO Ag

CELL INTERACTIONS IN PERIPHERAL LYMPHOID TISSUES Marginal zone Arteriole Germinal center T cell area – PALS paracortex DC – T cell contact DC Proliferating B cells centroblasts T B B – T cell contact Somatic hypermutation Additional gene rearrangement Isotype switch Plasma cell differentiation Antibody production Memory B cells

SELECTION OF HIGH AFFINITY B CELLS UPON INTERACTION WITH FOLLICULAR DENDRITIC CELLS Inhibition of apoptosis Tight junction B cell VLA-4 LFA-1 VCAM-1 ICAM-1 BCR CD21 C3d

INTERACTION OF ANTIGEN-SPECIFIC T AND B CELLS FDC CD40 Fas B cell differentiation apoptosis CD40L FasL GC T cell B and T cells recognize the same antigen

How antigen-specific Ab production is maintained? MODEL 2. MODEL 1. memory B cell plasma cell Bystander help: Cross-reactive antigens TLR ligands Cytokines... Memory B cells continuously differentiate into plasma cells Long-lived plasma cells in the bone marrow MODEL 3. Repeated activation with the antigen drives B cell activation and plasma cell differentiation role of follicular dendritic cells in antigen storage (months-years?) Polio: reinfections with Sabin vaccine strain subclinical infections (Diphteria in 10% of the population) hidden antigens (Measles genes persist in neurons – can induce Subacute Sclerosing Panencephalitis)

CELL INTERACTIONS IN THE PARACORTEX DC CD40 CD40L Antigen recognition by B and T lymphocytes T cell MHC TCR B7 CD28 B cell

RECEPTOR MEDIATED CELL ACTIVATION Conformational change Ligand Ligand Cross - linking Conformational change SIGNAL SIGNAL

CROSS – LINKING OF THE RECEPTOR INITIATES A SIGNALING CASCADE ligand kinase activation phosphorylation recruitment of adaptors SIGNAL Gene transcription Activation of transcription factors

THE IgM B-CELL RECEPTOR a a THE IgM B-CELL RECEPTOR antigen binding mIg molecule H L V b a Ig-a/Ig-b heterodimer Signal transduction Lyn Kinases Syk Btk SHP-1 Phosphatases SLP-65/BLNK PLC HS1 Vav Adaptors + substrates

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

RECENT MODEL OF B-CELL RECEPTOR MEDIATED SIGNALING Subsequent activation of 2 kinases 1. Cross-linking Ag Lyn 2. Src-family kinase activation P and ITAM phosphorylation 3. Syk recruitment and activation Syk 4. SLP phosphorylation + Ca release SLP P Calcium release P = ITAM

Main steps of B-cell signal transduction

Activation of B-cells by receptor crosslinking

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

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

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 1hr 6 hrs 12 hrs 24 hrs

ANTIBODY – METIDATED EFFECTOR FUNCTIONS Neutralization – binding of the antibody inhibits the adhesion of the pathogen, its entry or multipolication Opsonization – binding of antibodies induces complement activation and promotes binding to immun cells through complement (CR1) and immunoglobulin binding (FcR) receptors Antibody isotypes differ in their complement activating and FcR binding capabilities

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

Models of Human Rhinovirus 14 neutralised by monoclonal antibodies 30 strongly neutralising McAb 60 strongly neutralising McAb Fab regions Human Rhinovirus 14 - a common cold virus 30nm Models of Human Rhinovirus 14 neutralised by monoclonal antibodies 60 weakly neutralising McAb Fab regions

Electron micrographs of Antibodies and complement opsonising Epstein Barr Virus (EBV) Negatively stained EBV EBV coated with antibodies and activated complement components EBV coated with a corona of anti-EBV antibodies

T – CELLS PROMOTE B – CELL DIFFERENTIATION ANTIGEN CYTOKINES PLASMA CELL ISOTYPE SWITCH AND AFFINITY MATURATION OCCURS IN COLLABORATION WITH T – CELLS ONLY HOW T – CELLS RECOGNIZE ANTIGENS?