B CELL DEVELOPMENT IN THE BONE MARROW

ORDERED B-CELL DEVELOPMENT immature B cell pre B cell pro B cell ANTIGEN RECOGNIZING RECEPTOR H2L2 pro B cell H-chain + surrogate L-chain SIGNALING RECEPTOR NO ANTIGEN RECOGNIZING RECEPTOR

B B Stromal cell

Bone marrow stromal cells nurture developing B cells 1. Specific cell-cell contacts between stromal cells and developing B cells Cell-cell contact Secreted Factors - CYTOKINES 2. Secretion of cytokines by stromal cells B Stromal cell Types of cytokines and cell-cell contacts needed at each stage of differentiation are different

Cytokines and cell-cell contacts at each stage of differentiation are different D-J rearranged Stem Early pro-B c-Kit Receptor Tyrosine kinase Stem cell factor Cell-bound growth factor VLA-4 (Integrin) Cell adhesion molecules VCAM-1 (Ig superfamily) Stromal cell No Ig DJ rearrangement (H chain) L-chain genes in germline form

Cytokines and cell-cell contacts at each stage of differentiation are different V-D-J rearranged Light chain germline µ-chain made Interleukin-7 receptor Interleukin-7 Growth factor Early pro-B VLA-4 (Integrin) VCAM-1 (Ig superfamily) Late pro-B Pre-B Stromal cell

Ligand for the pre-B cell receptor is unknown CHm Heavy chain VHDHJH V-preB l5 Iga & Igb signal transduction molecules Transiently expressed when VHDHJH CHm is productively rearranged VpreB/l5 - the surrogate light chain (SLC), is required for surface expression Ligand for the pre-B cell receptor is unknown

Ligation of the pre-B cell receptor 1. Suppresses further H chain rearrangement 2. Triggers entry into cell cycle Large Pre-B Unknown ligand of pre-B cell receptor 1. Ensures only one specificty of Ab expressed per cell Stromal cell 2. Expands only the pre-B cells with in frame VHDHJH joins ALLELIC EXCLUSION

Ligation of the pre-B cell receptor triggers entry into the cell cycle Large pre-B 100X expansion Large Pre-B Many large pre-B cells with identical pre-B receptors Proliferation V-J light chain rearranged V-J light chain expression is quite efficient with an 85% success rate From a single µ chain 85 receptor!!! Large pre-B Y Immature B cell Light chain expressed IgM displayed on surface IgM Proliferation stops Pre-receptor not displayed Small pre-B Intracellular VDJCH chain VL-JL rearranges

B cell receptor Heavy chain VHDHJH Light chain VLJLCL CHm Iga & Igb signal transduction molecules L chain is rearranged

RECEPTOR EXPRESSION DURING B-CELL DEVELOPMENT

Allelic exclusion even though every B cell possesses a maternal and paternal locus of both genes, B cells express a single heavy and light chain. Does this „crippled” expression serve a purpose? Many of the genes (not all) are expressed co-dominantly, how could B cells manage to silence their other BCR-coding allels? ALLELIC EXCLUSION

Evidence for allelic exclusion ALLOTYPE- a polymorphism in the Heavy chain C region of Ig Allotypes can be identified by staining B cell surface Ig with antibodies a/a b/b a/b Y B a Y B b Y B a Y B b AND Y B a b Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two specificities of antibody per cell

Allelic exclusion is needed for efficient clonal selection Antibody S. typhi S. typhi All daughter cells must express the same Ig specificity otherwise the efficiency of the response would be compromised Suppression of H chain gene rearrangement helps to prevent the emergence of new daughter specificities during proliferation after clonal selection

Allelic exclusion prevents unwanted responses One Ag receptor per cell IF there were two Ag receptors per cell Y B Y Self antigen expressed by e.g. Liver cells B Y S. aureus S. aureus Y Anti S. aureus Antibodies Y Anti Liver cell Abs Y Anti S. aureus Antibodies Suppression of H chain gene rearrangement ensures only one specificty of Ab expressed per cell. Prevents induction of unwanted responses by pathogens

Allelic exclusion is needed to prevent holes in the repertoire Y B One specificity of Ag receptor per cell Y B IF there were two specificities of Ag receptor per cell Anti-brain Ig Anti-brain Ig AND anti-S. Aureus Ig Exclusion of anti-brain B cells i.e. self tolerance BUT anti S.Aureus B cells will be excluded leaving a “hole in the repertoire” B Deletion Anergy OR Y B S. aureus

THE RESULT OF SOMATIC GENE REARRANGEMENTS Combination of gene segments results in a huge number of various variable regions of the heavy and light chains expressed by different B-cells SOMATIC GENE REARRANGEMENT 2. Successful somatic rearrangement in one chromosome inhibits gene rearrangement in the other chromosome ALLELIC EXCLUSION 3. One B-cell produces only one type of heavy and one type of light chain COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE 4. The B-cell pool consist of B-cells with differently rearranged immunoglobulin genes INDEPENDENT OF ANTIGEN OCCURS DURING B-CELL DEVELOPMENT IN THE BONE MARROW

Allelic exclusion helps diagnose and monitor lymphoma: Due to clonal expansion of a single cell that contains a unique rearrangement the amount of cancer cells in blood or in bone marrow can be determined Can be used to monitor residual tumor cells upon treatment

Stages of B cell development Stem Cell Early pro-B cell Late pro-B cell Large pre-B cell Peripheral Y Small pre-B cell Immature B cell Mature B cell Receptor H+L Y Receptor H+L Each stage of development is defined by IgH and IgL chain genes, expression of adhesion molecules and cytokine receptors

SYNTHESIS OF IMMUNOGLOBULINS Secreted Ig Membrane Ig Golgi ER H and L chains are synthesized on separated ribosomes CHAPERONES Leader sequence Ribosome mRNA

DEVELOPMENT OF B-LYMPHOCYTES IN THE BONE MARROW Limphoid precursor B cells recognizing self structures Cell surface molecules MHC proteins Common molecules of haemopoetic cells apoptosis, clonal deletion Soluble molecules House keeping genes Metabolites functional unresponsiveness anergia c-kit/CD44 RAG-1/RAG-2 H rearrrangement H átrendeződés  Surrogate L L rearrangement B Selection clonal deletion B Other specificites B B PERIPHERAL LYMPHOID TISSUES

Negative selection of immature B-cells in the bone marrow Potential B-cell repertoire BONE MARROW Self structure Self recognition Clonal deletion RNA editing PERIPHERAL LYMPHOID ORGANS Available B-cell repertoire Foreign antigen independent Hozzáférhető sokféleség…. About 30 billion mature naive B cells leave the bone marrow per day to circulate in blood

Immature B cells with specificity for multivalent self antigens are retained in the bone marrow. Immature B cells that are not specific for a self antigen in bone marrow mature further to express IgM and IgD and leave the bone marrow (left panels). Immature B cells specific for a self antigen on bone marrow cells are retained in the bone marrow (right panels).

Receptor Editing of Immature B cells with self-reactive BCR (Bone Marrow)

Immature B cells specific for monovalent self antigens develop a state of anergy. Anergic B cells have a half life of 4-5 days (10% that of regular B cells)

How can mature B-cells express surface IgM and IgD

Co-Expression of cell surface IgM and IgG On Mature B-cells is controlled by alternative RNA processing

RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION Somatic rearrangement of Ig gene segments occurs in a highly controlled manner Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities In one individual a large B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains This potential B-cell repertoire is able to recognize a wide array of various antigens Immature B-cells express IgM and IgD surface Ig with the same variable domains

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

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.

„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-s bind immune complexes (Ag-Ab )  Ag detectable for 12 months following immunization  A single cell binds various antigens 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 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.

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