B Lymphocyte Development

Stages of B Cell Development –defined by the sequential rearrangement and expression of heavy- and light-chain immunoglobulin genes

(1) Stem Cell

(2) Pro-B Cell – progenitor cells with limited self-renewal capacity. Rearrangement of heavy-chain immunoglobulin gene segments takes place in these cells. No Igs on cell surface. (No expression)

(3) Pre-B Cell – VDJ_H joining leads to expression of a heavy chain on the surface of the cell. It combines with a surrogate light chain (the surrogate light chain takes the place of the real light chain for now. This permits the cell to divide further). (Expression of heavy, but not light) – L chain gene rearrangement)

(4) Immature (Naïve) B Cell – Real light chain made, the new heavy/light chain complex (IgM) replaces the Pre-B cell complex on the surface of the cell – rearrangement ceases

(5) Mature B Cell – has surface Ig, but has not yet encountered antigen

Antigen Independent Stage

B-cell development is driven by Allelic Exclusion (see definition below):

Pro-B Cell stage – the VDJ gene segments rearrange to form an H-chain (m ) on the first chromosome only

if this fails rearrangement, it is attempted on the second chromosome

if this too fails the cell dies; if it survives it proceeds to the Pre-B Cell stage.

Pre-B Cell stage – cell attempts to rearrange a k gene on the first chromosome (or which survived from the previous generation)

if this fails it attempts to do so on the second chromosome

if this fails it attempts to rearrange a l gene on the successful chromosome

if this fails it attempts to do so on the second chromsome

finally, if all fails the cell dies.

In summary – we just attempted to first build a heavy chain and then a light chain. A failure to do any of these steps results in B-cell death

recall that the pre-B cell expresses the m :k or m :l on its surface, with the light chains being surrogate

immature B-cell then produces real light chains ending up with the Immunoglobulin IgM on its surface

Most cell loss is due to failure to make heavy chains. This is because the opportunity for successive rearrangement attempts is much greater in the case of light-chain genes.

Ig gene rearrangement is closely coordinated with gene transcription

as rearrangement progresses proteins are transcribed that enhance the process of gene rearrangement by binding to extra promoters

Cell-surface expression of the products of rearranged immunoglobulin genes act as checkpoints of B-cell development

the heavy chain/surrogate light chain complex on pre-B cells and the heavy chain/light chain complex on immature B cells are used as checkpoints to tell the cell whether to procede or not

Important Terms relating to the Antigen Independent Stage of B-Cell Development:

VDJ Joining – splicing or recombination of DNA containing the Variable, Diversity, and Joining segments of the Ig genes.

Allelic Exclusion – if one allele of an Ig gene undergoes productive rearrangement, the other allele is blocked from undergoing rearrangement.

RAG Enzyme – Recombination Activation Gene products, consisting of two proteins, RAG1 and RAG2, which promote the VDJ joining process. Basically they run the whole rearrangement process. Without them you don’t get B-Cells.

TDT Enzyme – Terminal Deoxynucleotidyl Transferase, a DNA polymerase enzyme which adds nucleotides onto the 3’ end of DNA in a random template independent fashion; contributes to junctional diversity in the VDJ joining process. Therefore this enzyme increases complexity in heavy chain rearrangement.

CD79a (Iga), CD79b (Igb) – These are coreceptors that arrange around the Ig when it reaches the cell surface. They serve the function of transmitting signals intracellularly after something binds to the Ig. This is needed because the Ig can’t send signal on its own. These molecules are important in this process because they signal that the Ig properly reached the cell surface.

Antigen Dependent Stage

naïve B Cell leaves the bone marrow and floats around in the blood

by now the surrogate light chain containing complex has been removed and what we have is a IgM on the surface

clonal deletion – Any naïve (i.e. immature) B-cell whose IgM is able to recognize self molecules is eliminated at this point, assuring that no B-cells are produced that are self-reactive

the exception to this is when the cells are able to bind soluble self antigens. The cells are rendered unresponsive to these Ags and bear little surface IgM. They migrate to the periphery where they express IgD but remain unresponsive, and if in competition with other B cells in the periphery, they are rapidly lost.

After this, B-cells get into the lymph nodes and the spleen where they first encounter foreign antigen in the T-cell zone of the lymph nodes/spleen

Once the B-cells enter the lymph node, encounter Ag, and are activated by T_H2 cells one of two things happen:

(1) Some B cells migrate to medullary cords of the lymph nodes or to the bone marrow and complete their differentiation into antibody-secreting plasma cells and undergo Class Switching (see below)

(2) One or a few B cells migrate into a nearby follicle, forming a germinal center where rapid proliferation and somatic hyper-mutation occur

Affinity Maturation – somatic mutation generates B-cells with an even greater affinity for the previously encountered Ag

finally, B cell surface immunoglobulins bind antigen and internalize and make MHC II; T helper cell then binds to the MHC and secretes cytokines that stimulates the B cell to secrete Ig of that type (CD40:CD40L interaction occurs)

CD40/CD40 Ligand

B-cell growth is triggered in part by the binding of CD40 ligand expressed on activated helper T cells to CD40 on the B-cell surface
this complex proceeds to control Class Switching

Class Switching (isotype switching) – the process of changing the class of antibody secreted (IgM Þ IgG, IgA, IgE)

does not affect antibody specificity significantly, but alters the effector functions an antibody can engage

mechanism: part of the DNA coding for the constant region is spliced

(1) Permissive Signal (initiates class switching) – CD40/CD40L + cross linking to B-Cell Receptor

(2) Specific Signal (mediates/influences class switching) – mediated by cytokines: IL-4 Þ IgE, IgG4; TGFb Þ IgA

B-1 B Cells vs. B-2 B Cells

CD5 is expressed on B-1 B cells (self-renewing), not on B-2 B cells (conventional B-cells that are not self-renewing)

B-1 B cells are of interest to clinicians as they are the origin of common B-cell tumor in the disease chronic lymphocytic leukemia (CLL)

CLL cells often display CD5, which is a useful diagnostic clue.

Property	B-1 B cells	Conventional B-2 B cells
When 1^st produced	Fetus	After birth
Mode of renewal	Self-renewing	Replaced from bone marrow
Production of immunoglobulin	High	Low
Specificity	Degenerate	Precise
Isotypes secreted	IgM >> IgG	IgG > IgM
Somatic hypermutation	Low-none	High
Response to carbohydrate antigen	Yes	Maybe
Response to protein antigen	Maybe	Yes

B-Cell Diseases

Acute Lymphoblastic Leukemia (ALL) – Malignancy of B cell precursors. One of the most common malignancies in children.

Chronic Lymphocytic Leukemia (CLL) – Malignancy of mature, naïve B cells. One of the most common type of leukemia in older adults in the United States. Associated with CD5 (see above).

Immunoblastic Lymphoma – A rapidly growing malignancy of mature, activated B cells. One of the most common malignancies in AIDS patients

Multiple Myeloma – A malignancy of plasma cells, usually seen in older adults. A key clinical feature is high levels of monoclonal immunoglobulin protein in the serum.