Cluster Designation Age: >2 years age

Total T cells (CD3+)

Helper T-cells: % CD4+ and CD3+

Absolute CD4+ and CD3+

~70%

31-58443-1345

Suppressor T-cells: % CD8+ and CD3+

Absolute CD8+ and CD3+ 13-40171-914

Total T-cells: % CD3+

Absolute CD3+ 60-87782-2204

B-cells: % CD19+ and CD3-

Absolute CD19+ and CD3- 6-2362-540

NK-cells: % [CD16+/CD56+] and CD3-

Absolute [CD16+/CD56+] and CD3- 4-2660-590

CD4: CD8 ratio 0.8-3.5

adapted from http://www.aruplab.com/guides/ug/tests/0095892.htm

B CELL DEVELOPMENT AND ACTIVATION

• In healthy people, there are mature B cells with the capacity to make antibodies to virtually any antigen.

• Bone marrow is the primary lymphoid organ in which B cell development occurs.

• Bone marrow is the primary lymphoid organ in which B cell development occurs.

• Following initial development in bone marrow, mature B cells migrate to various secondary lymphoid tissues, including lymph nodes, spleen, gut-associated lymphoid tissue and blood.

• There, mature B cells can interact with antigen, become activated, and further differentiate into antibody-secreting cells

• B and T cells undergo distinct differentiation pathways.

• B cells are generated in the bone marrow, with mature B cells, which are ready to respond to antigen, then exiting and migrating to lymph nodes and spleen. T cells are generated in the thymus (more on this tomorrow).

• The development of B cells, starting from hematopoietic stem cells and ending with cells that produce antibodies, can be divided into four phases:

 

Phase 1 – development of B cells in bone marrow

• This first phase of B cell development is the generation of B cells in bone marrow.

• There, stem cells develop into pro-B cells, then pre-B cells, and finally mature B cells, which exit the bone marrow and migrate to secondary lymphoid organs.

• This phase of B cell development is not driven by contact with antigen: antigen independent.

• The DNA rearrangements that result in a functional cell-surface immunoglobulin molecule occur during this phase.

  

Phase 1 – development of B cells in bone marrow

• Stem cells have both their H-chain and L-chain genes in germ-line, un-rearranged configuration

• The earliest cell that has made the commitment to the B cell lineage is the pro-B cell: pro-B cells have begun to rearrange their H-chain gene.

• Once a B lineage cell expresses cell surface H-chain () it is defined as a pre-B cell.

• However, the early pre-B cell receptor is not the final form of surface immunoglobulin: H-chain + surrogate light chain (molecule that mimics L-chain)

• Further DNA rearrangements result in the formation of a functional L-chain, then IgM (H-chain + L-chain) is expressed on the cell surface.

• When a cell has productive rearrangements of both H- and L-chains) it becomes an immature B cell:

• This first phase of B cell development in bone marrow is dependent on association with stromal cells.

• Stromal cells are non-lymphoid cells that provide an appropriate microenvironment for B cell development.

• Bone marrow stromal cells produce both cell surface-stimulatory molecules, as well as growth factors and cytokines, which help drive B cell development.

• For a B cell to survive this phase of development, it must have productive rearrangements of both H-chain and L-chain.

• Failure to do this results in cell death - cells that have unproductive rearrangements (such as rearrangements that are not in a correct reading frame) are eliminated.

• A given B cell can undergo repeated rearrangements.

• The rearrangements that result in functional H- and L-chains occur in a specific order:

• Expression of a functional B cell receptor protein on the cell surface stops further rearrangement of the gene encoding that product:

Phase 2 – elimination of self-reactive cells

• Once B cells express a functional cell surface receptor for antigen (immature/mature B cell stage) they have the potential to be stimulated by contact with antigen, and to become antibody-secreting cells.

• Since the DNA rearrangements that result in functional H-chain and L-chain are not antigen-driven, a fraction of immature B cells will have a BCR that, by chance, reacts with some component of self - self antigen reactive cells

• These immature B cells are removed by clonal deletion, either in the bone marrow, or shortly after leaving the bone marrow.

 

• Encounter with self-antigen results in apoptosis (death), or anergy (unresponsiveness)

• B cells that survive this step express surface IgD as well as IgM: mature B cells

• After elimination of self-reactive B cells, mature cells, which express both cell surface IgM and IgD, are ready to leave the bone marrow, can interact with antigen in secondary lymphoid organs.

• How do these immature B cells know that the antigen that they’re binding is a self Ag? They don’t:– B cells are more susceptible to anergy at this stage in

development– They become anergic if they don’t receive a co-

stimulatory signal:

• Therefore, signaling via the BCR alone is not only not sufficient to activate the B cell – it can induce apoptosis or anergy

• Second signals (co-stimulatory signals) are necessary for activation.

Phase 2 – elimination of self-reactive B cells, generation of mature IgM+, IgD+ cells:

Phase 3 – activation of B cells on contact with antigen

• Following the generation of a functional B cell receptor for Ag, and the removal of self-reactive cells, mature Ag-responsive B cells (IgM+, IgD+) emigrate from bone marrow.

• These mature B cells go to secondary lymphoid organs.

 

• In the lymph node, B cells gather in primary lymphoid follicles, where they receive viability-promoting signals, interacting with follicular dendritic cells, and wait for antigen

• B cells enter lymph nodes via high endothelial venules (HEV) to reach these primary follicles.

• B cells can recirculate out via the lymphatic circulation, and back into blood.

 

• Antigen-specific B cells are detained in the T cell-areas, where they interact with antigen, and with antigen-specific activated helper T cells. Stimulated antigen-specific B cells then proliferate and differentiate, eventually forming plasma cells and germinal centers:

 

• Binding of antigen to the B cell’s antigen receptor results in an initial activation signal (first signal) if there is receptor cross-linking.

• Cross-linking of the B cell receptor results in a cascade of intracellular signals, which results in the induction of specific gene expression leading to cellular proliferation and differentiation.

• However, signaling via the BCR alone is not sufficient to activate the B cell -

• Second signals (co-stimulatory signals) are necessary for activation.

Laâbi, Y. and A. Strasser. Science 289:883, 2000

activation signal

activation signal

Phase 4 – differentiation to antibody-secreting cells

• Some of the progeny of these antigen-activated B cells differentiate into IgM-secreting plasma cells (antibody-secreting cells).

 

• Plasma cells:– terminally-differentiated cells– derived from activated B cells or memory

cells– loaded with endoplasmic reticulum– devoted to protein (antibody) synthesis– no longer express surface immunoglobulin

or MHC class II– no longer responsive to antigen contact– live for several weeks– migrate away from the site of initial contact

with helper T cells, either to the medullary cords of the lymph nodes or to the bone marrow 

• Other antigen activated B cells give rise to germinal centers (GC), zones of proliferating activated B cells:

 

• These germinal centers (GC) contains:– proliferating (D - centroblasts) B cells – differentiating (L - centrocytes) B cells

• B cells can interact with an antigen that is bound to the surface of follicular dendritic cells in the lymph nodes.

• These cells trap and concentrate antigen, maximizing the interaction of antigen with B cells

 

• Germinal centers are where isotype switching and somatic hypermutation occur.  • Somatic hypermutation:

– rapid mutation (hypermutation) of immunoglobulin genes

– results in antigen-binding affinity that is higher, or lower, than its original binding affinity

– selection by antigen results in the generation of BCR with increased affinity for antigen

 

• Only those B cells that have enhanced their antigen receptor’s binding affinity survive.

• This selection process involves competition for both antigen and for helper T cells.

• Antigen is trapped on the surface of follicular dendritic cells in the form of immune complexes (antigen + antibody complexes).

• B cells that bind to Ag with high affinity live, others die by apoptosis.

• Centrocytes interact with T cells by presenting processed antigen to them via their MHC class II molecules.

• Centrocytes that receive co-signaling (via CD40, MHC class II and cytokines), as well as signaling via their antigen receptor survive

• Centrocytes that do not bind antigen and T cells with sufficient affinity die by apoptosis.

• These germinal centers (GC) contains:– proliferating (D - centroblasts) B cells – differentiating (L - centrocytes) B cells

• Isotype switching - change in the type of H-chain that is used by a given B cell, also occurs in germinal centers:

• Isotype switching involves DNA rearrangements - replacement of one H-chain class gene with another

• Isotype switching also occurs in germinal centers

• Isotype switching is guided by the pattern of cytokines that are produced by helper T cells:

• These somatically mutated and isotype switched B cells can then continue to differentiate into memory cells or plasma cells, producing:

– IgG or other switched isotypes

– much higher affinity Ab, due to somatic hypermutation increasing the antigen binding affinity

– migrate from the secondary lymphoid organs to the bone marrow, where cytokines (IL-6, IL-11) produced by bone marrow stromal cells keep these cells viable and producing Abs

Together, these processes result in a 1-2 log increase in the number of antigen-specific B cells (clonal selection and expansion), an increase in antibody binding affinity for antigen (somatic hypermutation), and the expression of new Ig subclasses (Ig isotype switching):

• Other germinal center B cells develop into memory B cells:– quiescent, long-lived– increased in frequency following primary responses– found in secondary lymphoid organs (spleen, lymph nodes,

Peyer’s patch)– posses high-affinity, isotype-switched (IgG, IgA, IgE)

antigen receptors– form a pool of cells ready to mount a rapid secondary

antibody response, on subsequent exposure to antigen:  

• The combined result of clonal selection (expansion of pool size of Ag-reactive cells), somatic hypermutation, isotype switching, and the generation of memory cells, is the creation of a pool of cells that can respond rapidly and vigorously to subsequent contact with antigen, with high-affinity, IgG and IgA antibodies.

 

B cell cancers mirror these different stages of B cell development