B Cell Development
B Lymphocyte Development
There are many stages of the development of precursor B cells into mature B lymphocytes
B cells develop from pluripotent hematopoietic stem cells within primary lymphoid organs
 Bone marrow (rodents, primates)
 Peyer’s patches (ruminants, pigs, rabbits)

Bursa of fabricius (birds)
B cell developmental stages are based on a mouse model (details should vary with species)
 Pro-B cell o Earliest distinguishable cell in the B cell lineage o Has undergone D-J segment heavy chain rearrangement o Does not yet produce antibody
 Pre-B cell o Has undergone complete V-D-J segment heavy chain rearrangement o Rearranged V-D-J unit is placed next to the Cμ gene

Pre-B cell produces only μ heavy chain polypeptide (cannot produce light chains)
 The μ heavy chain is paired with a surrogate light chain to form the pre-BCR
Surrogate light chain is the product of two non-rearranging genes
 Lambda 5 (λ5)

VpreB
Pre-BCR is expressed on the cell surface
Pre-BCR is associated with an Igα/Igβ heterodimer
B cell development cannot continue without signaling via the pre-BCR

Immature B cell o Has undergone a complete V-J light chain rearrangement (V
L
will be of κ or λ type) o Light chains ( not surrogate light chains ) are paired with the μ heavy chain polypeptides o Consequently, immature B cell expresses IgM (with full heavy and light chains) at cell surface o Recognition of antigen by membrane IgM can have different consequences

Clonal deletion (death of immature B cell by apoptosis)

Anergy (long-term inactivation (not death) of immature B cell)

Receptor editing
-
Antibody L chain genes undergo V(D)J recombination using unrearranged V or J elements to generate an IgM molecule with new antigen specificity

These processes guarantee that self-reactive immature B cells are either removed,
turned off, or modified in order to maximize future immune response potential

Mature B cell o Have both IgM and IgD at cell surfaces

Antigen specificities of these antibodies are identical due to alternative splicing o Mature B cells migrate to secondary lymphoid organs in order to seek out foreign antigen o Activation by foreign antigen causes further B cell differentiation into plasma or memory cells

Plasma cell o Product of B cell activation

Interaction of mature B cells with antigen causes proliferation and differentiation o Specialized cell that synthesizes and secretes large amounts of antibody molecules

Antibodies are of IgM class during early immune response o Processes that occur after initial immune response

Class (isotype) switching
Occurs if B cell receives cytokines from T cells
-
Result is some progeny of B cell clonal expansion express IgG, IgG, or IgA
Light chain does not undergo switching

Somatic hypermutation
Production of B cells whose antibody variable region genes can synthesize antibodies with higher affinities to the activating antigen (affinity maturation)

 Memory cell o Long-lived product of mature B cell clonal expansion o Does not secrete antibody o Remains dormant until subsequent infection by antigen (secondary response)

Undergoes class switching to express IgG, IgE, or IgA on cell surface
Will secrete respective antibody if triggered by antigen

Results in larger and more rapid immune response
B cell receptor (BCR)
 Composed of membrane Ig and two closely-associated transmembrane molecules o Igα, Igβ

Linked together by a S-S bond to form a heterodimer


Do not bind antigen
Act as signal transduction molecules
-
Transmit signal to B cell after antigen binds to membrane Ig
Applications of Antibodies
Basic research (virtually every field of biological science utilizes antibodies in experiments)
Diagnostic

Antibodies can be used to detect extremely small concentrations of substances (i.e. hormones) in biologic fluids such as urine, blood, and tissue sections
Vaccination

Eliciting specific antibodies via artificial vaccin. was the 1 st major medical application of immunology
Therapeutic

The use of antibodies and antibody-based reagents can be used to treat disease o ~30% of all biological proteins currently in human clinical trials are GE antibodies
Antibody Sources
Serum antibodies

Acquired via immunization of animals (collection of antibodies made in response to vaccination)
 Immune serum is known as antiserum

Antibodies from antiserum are polyclonal antibodies because antiserum contains a heterogeneous collection of antibodies derived from various B cell clones o These antibodies will include those specific for many different epitopes on the immunogen
 Serum antibodies can be extremely specific and of high affinity
 No two antiserum preparations are identical, making it difficult to standardize their applications
Monoclonal antibodies (MAbs)
 Produced from hybridomas o Formed by fusing immortal (cancer) cells with antibody-making B cells o Can produce an unlimited supply of antibodies

Allows for the wide availability of standardized antibody reagents
 MAbs are typically produced in mice, so the use of MAbs in humans requires the replacement of mouse protein sequences with human antibody sequences o Chimeric antibodies

Have mouse variable regions and human constant regions o Humanized antibodies

CDR regions of antigen-specific mouse antibody “grafted” into human Ig sequence o Xenomice
 Transgenic technology is used to construct “xenomice” that only produce human
MAbs (human heavy and light chain Ig loci inserted into the mouse genome)
Immunoassays
Enzyme-linked immunosorbent assay

Uses antibodies coupled to enzymes to detect substances in solution (i.e. ELISA)
Immunofluorescence assay

Antibodies coupled with fluorochromes are used to detect antigens in tissue sections or cells
Flow cytometry

 Cells “labeled” by antibodies coupled with fluorochromes are detected with a flow cytometer