TODAY B CELL DEVELOPMENT

STAGES OF B CELL DEVELOPMENT 1. B Cells develop from pluripotent stem cells in the bone marrow. 1 GENERATIVE LYMPHOID ORGANS 2 2. The stem cell develops into a mature IgM+ B cell. The initial steps in B cell development occur in the bone marrow. PERIPHERAL LYMPHOID ORGANS

B Lymphocytes are predicted to generate approximately 1 X 1011 distinct antigen receptors. However, the human genome only contains about 35,000 distinct genes. How does such a limited genome enable the generation of an almost infinite number of antigen receptors?

Answer: Diversity is generated through somatic recombination of gene segments that code for the variable regions of the receptors.

Going from DNA (gene) to protein: the usual way. Genomic DNA Promoter Exon 1 Exon 2 Primary Transcript Transcription mRNA Splicing Typically, a gene is organized into a distinct linear arrangement of introns and exons that encode distinct gene products. Protein Translation

THE GERMLINE ORGANIZATION OF ANTIGEN RECEPTOR GENE LOCI. The Ig Heavy chain locus contains up to a few hundred variable (V) region genes. The variable region gene segments are followed by diversity (D) and joining (J) gene segments. The heavy and light chain loci are completely fragmented. They consist of hundreds of individual gene fragments that must be combined in the correct order to generate a single BCR. The Light chain loci displays a similar organization to the Heavy chain locus, except that it lacks any D gene segments. There are two light chain loci - k and l.

GENERATING DIVERSITY BY V(D)J SOMATIC RECOMBINATION 3. A V gene segment then recombines with the D-J segment, generating a recombined V-D-J gene. 4. The new gene is then transcribed. 5. The V-D-J gene segment is spliced to the first C region RNA (m Chain) 1. V, D and J gene segments are arranged in tandem. 2. In a single lymphocyte clone, one D gene segment randomly recombines with one J gene segment. 6. The resulting mRNA is translated to generate a unique IgM heavy chain. Generation of a variable gene segment occurs through the random recombination of V,D and J gene segments, thereby ensuring that each lymphocyte endcodes a distinct BCR with a distinct antigen specificity.

V D J constant VDJ recombination #1 variable constant VDJ recombination #2 variable constant VARIABLE REGION While each VDJ recombination yields a unique antigen specificity, the constant region remains fixed, thereby ensuring conserved effector functions. 1. Each recombination generates a unique variable region - unique antigen specificity. 2. In contrast, the constant region does not change - conserved effector function.

SOURCES OF DIVERSITY

STAGES OF B CELL DEVELOPMENT 1. B Cells develop from pluripotent stem cells in the bone marrow. 1 GENERATIVE LYMPHOID ORGANS 2 2. The stem cell develops into a mature IgM+ B cell. 3 3. The mature, naïve B cells circulates through the peripheral lymphoid organs. PERIPHERAL LYMPHOID ORGANS

STAGES OF B CELL DEVELOPMENT 1. B Cells develop from pluripotent stem cells in the bone marrow. 1 GENERATIVE LYMPHOID ORGANS 2 2. The stem cell develops into a mature IgM+ B cell. 3 3. The mature, naïve B cells circulates through the peripheral lymphoid organs. PERIPHERAL LYMPHOID ORGANS 4. B cells that fail to encounter antigen die through apoptosis. 4

STAGES OF B CELL DEVELOPMENT 5 5. B cells that encounter antigen are activated. PERIPHERAL LYMPHOID ORGANS

STAGES OF B CELL ACTIVATION 2. Helper T cells stimulate B cells. 1. Naïve B cell encounters antigen.

PRESENTATION OF ANTIGENS TO T HELPER CELLS BY B CELLS 4. B cell stimulates T cell activation. 3. Antigen is processed and presented on a Class II MHC molecules. (B cell acts as classical APC) 2. The receptor triggers endocytosis of the antigen. 1. The naïve B cell receptor binds its cognate antigen. The B cell can act as an APC for CD4 T cells. Note that the BCR is the receptor for internalization of the microbial antigen. This means that the B cell will only activate T cells that respond against the same microbial antigen. Thus, the responding immune cells (both B and T) retain a specificity for the antigen.

HELPER T CELL ACTIVATION OF B CELLS 2. T cells commence production of molecules that activate B cells (CD40, cytokines). 1. B Cells process and present antigens to helper T cells. 4. T helper cell signals induce B cell activation (proliferation and differentiation). 3. The combination of CD40 (physical contact) and cytokines activates B cells. The B cells activates the CD4 T cell, which responds by fully activating the B cell. The active B cell initiates clonal expansion and starts to secrete antibodies. The only B cells to expand commence antibody production are ones with the same antigen specificity as the activating CD4 T cell. This means that the immune response remains restricted to the foreign antigen. All B cells and T cells that do not express receptors with the correct antigen specificity are essentially blind to any ongoing infection.

STAGES OF B CELL ACTIVATION 3. Activated B cells begin clonal expansion. 2. Helper T cells stimulate B cells. 1. Naïve B cell encounters antigen.

CLONAL EXPANSION Each B and T cell encodes a unique BCR or TCR with a unique specificity. When a BCR or TCR encounters its cognate antigen the B cell or T cell is stimulated to undergo multiple rounds of cell division, thereby expanding. Each daughter is identical to the parent cell, i.e. a clone. Notice how clonal expansion is driven by a microbial antigen. This means that as soon as the immune response is complete and the antigen is eliminated there are no antigens to sustain clonal expansion. In the absence of a stimulating antigen, the B expanded B cell population collapses (driven by apoptosis). Essentially, this means that the levels of antigens dictate the levels of the responding B cells. Lots of microbial antigen = lots of B cell clonal expansion = powerful immune response. Successful immune response = loss of microbial antigens = apoptosis of responding B cells = reduction in B cell population.

STAGES OF B CELL ACTIVATION 3. Activated B cells begin clonal expansion. 4. Some B cells differentiate into antibody secreting plasma cells. 2. Helper T cells stimulate B cells. 1. Naïve B cell encounters antigen.

STAGES OF B CELL ACTIVATION 3. Activated B cells begin clonal expansion. 5. Others become memory cells. 4. Some B cells differentiate into antibody secreting plasma cells. 2. Helper T cells stimulate B cells. 1. Naïve B cell encounters antigen.

MEMORY LYMPHOCYTES Memory Lymphocyte Some active lymphocytes differentiate into memory lymphocytes. Active Lymphocyte MEMORY LYMPHOCYTES: Produced from naïve lymphocytes as a result of antigen exposure. Persist for years in a quiescent state. Rapidly reactivated by repeat exposure to antigen and mediate faster, more potent immune responses.

Secondary Versus Primary Antibody Responses. THE SECONDARY RESPONSE IS……. FASTER STRONGER MORE SPECIFIC

STAGES OF B CELL ACTIVATION 3. Activated B cells begin clonal expansion. 6. Others switch class to produce a new Ig Isotype. 5. Others become memory cells. 4. Some B cells differentiate into antibody secreting plasma cells. 1. Naïve B cell encounters antigen. 2. Helper T cells stimulate B cells.

CLASS SWITCHING A clone of B cells is not committed to make a single Ig isotype forever. All mature B cells start out making IgM V d m g a e IgM mRNA IgM

CLASS SWITCHING A clone of B cells is not committed to make a single Ig isotype forever. V d m g a e B cell activation triggers rearrangements at the Ig locus g a e V Exposed DNA ends are joined together d m DNA encoding some constant regions is deleted End result: New class of Ig IgG

CLASS SWITCHING - AFFINITY A clone of B cells is not committed to make a single Ig isotype forever. V m d g a e Class switching does not affect the variable region. UNCHANGED V g a e IgM mRNA IgG mRNA IgG IgM Upon class switching the constant region (effector functions) changes, not the variable region (antigen binding). This means that antigen specificity is not altered by class switching events. The Antibody retains Antigen specificity throughout.

CLASS SWITCHING Class switching explains why IgM is not seen upon re-exposure to antigen. Ag 1. Mature B lymphocyte express IgM as BCR BCR IgM BCR Ag 2. Lymphocyte is activated by antigen and starts to secrete IgM IgG BCR Ag 3. Activation induces class switching, so that repeat exposures induce IgG, IgA or IgE.