chapter 6 immunology

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Chapter 6

The Development Of B-lymphocytes

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Chapter 6

1. The development of B cells in the bone marrow-1st of the 4 “broad stages” in the life cycle of B cells

2. Selection and further development of the B-cell repertoire

-2nd, 3rd and 4th stages in the life cycle of B cells

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The Development Of B Lymphocytes

1st stage = maturation in the bone marrow (primary lymphoid tissue) Acquires functional B-cell receptors -Ig gene rearrangements

2nd stage = testing of Ig (B-cell receptor) to normal constituents of the body (self-reactive) Potential for auto reactivity and autoimmune disease

3rd stage = a small fraction of immature B-cells become mature cells in 2 lymphoid tissues

4th stage = recirculation of mature B-cells between the blood, lymph and secondary lymphoid tissues.

5th stage = antigen contact B cell progeny (clonal expansion)

6th stage = differentiate into plasma cells and long-lived memory B cells

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The Development Of B Cells Can Be Divided Into six Broad Phases

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B Cells Develop In Bone Marrow And Then Migrate To Secondary Lymphoid Tissues

Immature, naïve B cells leave the bone marrow blood lymph nodes, the spleen, Peyer's patches and other 2 lymphoid tissues such as those lining the respiratory tract and finish their maturation into Mature, Naïve B-cells.

Blood, lymph nodes, spleen, Peyer’s patches2 lymphoid tissues

Bone marrow1 lymphoid tissues

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The Development Of B Cells In The Bone Marrow

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The Development Of B Cells In The Bone Marrow

Stages of B-cell development in the bone marrow Marked by successive steps in the rearrangement and expression of Ig

genes Gene rearrangement (somatic recombination) is controlled at each

step to produce a mature , naïve B cell Expressing immunoglobulin of a single antigen specificity

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B-cell Development In The Bone Marrow Proceeds Through Several Stages

Stem cell - Ig genes are in the germline configuration 1st rearrangements are the Ig heavy chain genes

Join D to J (defines the early pro-B cell) Early pro-B cell late pro-B cell

Join V to DJ Late pro-B large pre-B cell

Expression of a functional chain at the cell surface as part of the pre-B receptor

Large pre-B cells proliferate, producing small pre-B cells

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B-cell Development In The Bone Marrow Proceeds Through Several Stages

Large pre-B small pre-B cells Associated with the rearrangement of the Ig light chain

Successful light chain gene rearrangement and expression of IgM on the cell surface defines the immature B cell

Immature B cell mature B cell Mature B cell = use of alternative splicing of heavy-chain

mRNA to place IgD on the cell surface with IgM

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B Cell Development Proceeds Through Stages Defined By The Rearrangement & Expression Of The Ig Genes

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Pre-B-Cell Receptor

Large pre-B-cells are distinguished by a protein complex = pre-B-cell receptor1. Consists of heavy chain

2. “Surrogate” light chains

1. Made only in pre-B cells

3. Ig and Ig polypeptides

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The pre-B-cell Receptor Resembles The B-cell Receptor Except For The Surrogate Light

Chain

Note: although some pre-B-cell receptor molecules are on the cell surface, most are retained in the ER

B-cell receptor molecules lead to: Intracellular signals that halt

rearrangement at Ig heavy-chain locus

Synthesis of surrogate light chains

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Small pre-B-cells

Large pre-B cells proliferates producing many small pre-B cells

Small pre-B-cells No longer have pre-B-cell receptor Ig heavy chains are restricted to the cytoplasm Rearrangement of the Ig light-chain loci begins

Ig light-chains assemble with chains IgM transported to cell surface as a “functional” B-cell receptor complex B-cell is expressing IgM only = immature B cell

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Up To This Stage Development Of B Cells Occurs In The Bone Marrow And Does Not Require Interaction With

Specific Ag

Randomness in gene rearrangement results in some B-cell receptors being reactive against self components Unless removed from the B cell repertoire autoimmune

response autoimmune disease At this point immature B cells:

Will undergo selection for tolerance of normal constituents of the body

Will enter the peripheral circulation secondary lymphoid organ to finish maturation

Alternative mRNA splicing of Ig heavy-chain gene transcripts produces IgD (as well as IgM as membrane-bound Ig)

B-cell is now a mature B cell

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The Early Stages Of B-cell Development Are Dependent On Bone Marrow Stromal Cells

Bone marrow stromal cells provide specialize environment for B cells at various stages of maturation

1. Make specific cell-surface contacts with B cells Stem cells and early pro-B cells use the integrin VLA-4 to

bind to the adhesion molecule VCAM-1 on stromal cells

2. Produce growth factors that act on bound B cells Interactions between other cell adhesion molecules (cams)

promote the binding of the receptor Kit on the B cell to stem-cell factor (SCF) on the stromal cell

Activation of Kit causes the B cell to proliferate B cells at later stage of maturation require interleukin-7 (IL-

7) to stimulate their growth and proliferation

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Interactions Of Developing B Cells With Bone Marrow Stromal Cells

S

L

S

Light micrograph Electron micrograph

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The Survival Of A Developing B Cell Depends On The Productive Rearrangement Of A Heavy- & A Light-chain

Gene

Imprecision in gene rearrangement (including random addition of N and P nucleotides at gene segment joints) can result in a sequence with a reading frame that cannot be translated into an Ig chain (has a stop codon) Unproductive rearrangements = gene rearrangement

that cannot be translated into a protein Productive rearrangements = gene rearrangement that

preserve a correct reading frame and can be translated into a complete and functional Ig chain

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Every B Cell Has Two Copies Of Each Ig Loci (Heavy-chain Locus, The Light-chain Locus And The Light-chain Locus)

The two copies are on homologous chromosomes; one inherited from mother and one from father

Gene rearrangements can be made on both homologous chromosomes Unproductive rearrangement on one chromosome leads

to rearrangement at the locus on the other chromosome Productive rearrangements proceed to the next stage

of development If all rearrangements are unproductive B cell does

not produce Ig and dies in the bone marrow

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If one allele rearranges non-productively, then the B cell rearranges the other allele. If this also fails and no rearranged heavy or light chain results the cell will die.

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The Order Of Gene Rearrangements Leading To The Expression Of Cell-surface Ig

Occurs concurrently on two copies of H-chain loci.

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About half of B cells can make functional heavy chain

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5-6 rounds of cell division.

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Less than half of the B cells produce a functional antibody.

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Unproductive L-chain Gene Rearrangements Can Be Replaced By Further Gene Rearrangement

L-chain loci organization allows further rearrangement after an unproductive rearrangement

After an unproductive rearrangement of V to a J, a second rearrangement can be made by V2 or any other V that is on the 5’ side of the first joint, with a J that is on the 3’ side of the first joint

When the second joint is made, the intervening DNA containing the first joint will be excised

There are 5 J gene segments and many more V gene segments five successive attempts at productive rearrangement of the L-chain gene on a single chromosome

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Unproductive L-chain Gene Rearrangements Can Be Replaced By Further Gene Rearrangement

Rescue is shown for the light-chain gene

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Expression Of The Products Of Ig Genes Prevents Further Gene Rearrangement

Mechanisms ensure that B cell can make only one type of heavy chain and one type of light-chain

Successful gene rearrangement is signaled by the appearance of the protein product of the gene at the cell surface

A signal is sent back to the cell interior to shut down the processes of DNA recombination and repair needed for gene arrangement

RAG genes are turned off and no further rearrangement is possible Once heavy-chain gene is successfully rearranged also further

rearrangement of heavy-chain genes is shut down (Same process happens with light-chain)

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Signaling Via The Protein Product Is Used To Terminate Ig-gene Rearrangement

1. In pro-B cells, heavy chains assemble into dimers in the ER

• In the absence of light chains, heavy-chains assembled into a complex with the 5 and the VpreB polypeptides to form a surrogate L-chain and with Ig and Ig

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The Proteins Involved In Immunoglobulin-gene Rearrangement Are Controlled Developmentally

Each surrogate L-chain is formed from two proteins coded by genes away from the Ig loci: 5 substitutes for a

L-chain constant region and

VpreB substitutes for a V region

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2. The pre-B cell receptor (Pre-BCR) is on the surface for a short time and represents a checkpoint for B cell development

• Pre-BCR interacts unknown ligand to confirm that a functional heavy chain has been made and stop rearranging heavy chain.

• Pre-BCR is a positive signal that prevents apoptosis and allows B cell to start dividing.

• After checkpoint, pre-BCR is no longer made, heavy chain, Ig and Ig continue to be made in ER

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3. Proliferation of the large pre-B cell yields small pre-B cells that commence Ig light-chain rearrangement

• RAG genes (turned off in dividing pre-B cells) is turned on and light chain rearrangement begins

• On completion of a productive light chain gene rearrangement a light chain protein is made and assembles with heavy chain to form IgM

• IgM (BCR) associates with Ig and Ig and is transported to the cell surface

• Second Checkpoint- Presence of the BCR with Ig and Ig tells the cell to halt further light-chain gene rearrangements

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Ig Gene Rearrangement Steps Are Accompanied By Changes In The Other Protein Expression

The rearrangement of Ig genes and the expression of pre-BCR and IgM on the cell surface requires several categories of specialized proteins at different times during B-cell development

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Many B-cell Tumors Carry Chromosomal Translocations That Join Immunoglobulin Genes To Genes Regulating Cell Growth

Normal process as B cells cut, splice and mutate their Ig genes can cause mutations that produce a tumor cell

In B-cell tumors disruption of regulated growth is associated with aberrant Ig-gene rearrangement Joining an Ig gene to a gene (involved in the control of cellular

growth on a different chromosome) - translocation Proto-oncogenes, genes that cause cancer when their

function or expression is perturbed Viral genes responsible for transformation = oncogenes

and it was sometime before it was know they had evolved from cellular genes that control cell growth, division and differentiation

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Chromosomal Rearrangements In Burkitt’s Lymphoma

Parts of chromosome 8 and chromosome14 have been exchanged The sites of breakage and rejoining are in the proto-

oncogene MYC on chr8 and the Ig H-chain gene on chr14 or the or light chains genes on chr2 or chr22

MYC is normally involved in regulating cell division. Abnormal expression as a result of translocation causes increased growth

Another translocation frequently found is the fusion of an Ig gene to the proto-oncogene BCL-2

Translocations probably occur during the first attempt to rearrange a heavy-chain gene This would have counted as an unproductive

rearrangement and the other gene would then be rearranged

In cases where the 2nd rearrangement is also unproductive, the cell dies and thus cannot give rise to a tumor

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Chromosomal Rearrangements In Burkitt’s

Lymphoma

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B Cells Bearing CD5 Express A Distinctive Repertoire Of Receptors

Not all B cells conform to the developmental pathway Subset of human B cells

Arise early in embryonic development Expresses CD5

Cell surface glycoprotein marker for the human T-cell lineage

Minority subset of B cells (b-1-cells) precedes that of the majority subset (b-2-cells)

B-1-cells Little or no IgD on surface Distinctive repertoire of Ag receptors Know as CD5 B cells (CD5 is not essential for their function) Dominant B cells in pleural and peritoneal cavities 5% of B cells in humans Arises from a stem cell most active in prenatal period

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Ig H-chain Gene Rearrangements Use The VH Gene Segments Closest To The D Gene Segments

TdT is not expressed in early prenatal period, therefore H-chain gene of B-1 cells lack N nucleotides and their VDJ junctions are less diverse

Ab secreted by B-1 cells to be low affinity, but each binds to many different antigens (polyspecificity) Abs made against bacterial polysaccharides & carbohydrate

Ags but little against protein Ags Postnatal B-1 cells use more diverse repertoire of V gene segments,

and have abundant N nucleotides With time, B-1 cells no longer produced by the bone marrow Adult population of B-1 cells is maintained by the division of

existing B-1 cells at site in peripheral circulation Self-renewal dependent on the cytokine IL-10

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Comparison Of The Properties Of B-1 Cells And B-2 Cells

B-1 cells develop in the omentum and in the liver in the fetus

Produced by the bone marrow for only a short period around birth

Pool of self-renewing B-1 cells established that do not require bone marrow for survival

The limited diversity and low affinity of the antibodies made by B-1 cells suggest these are components of a simpler, less adaptive immune response

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Selection And Further Development Of The B-cell Repertoire

Mechanisms That Eliminate Or Inactivate Potentially

Autoreactive B Cells Now Come Into Action

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Self-reactive Immature B Cells Are Altered, Eliminated, Or Inactivated By Contact With Self-

antigens

A B cell first expresses IgM on its surface as an immature B cell

Mature B cell involves emigration form the bone marrow and the use of alternative splicing of H-chain mRNA to place IgD with IgM on the cell surface

Quality control mechanisms prevent the maturation of B cells whose receptors bind normal components of the human body – called self-antigens (protein, carbohydrates, lipids found on the surface of human cells)

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Binding To self-Ag In The Bone Marrow Can Lead To The Deletion Or Inactivation Of Immature B Cells

Immature B cells that do not encounter a self-antigen leave the bone marrow and enter the peripheral circulation expressing both IgM and IgD on their surface

When immature B cells express receptors that recognize common cell-surface components (multivalent self-antigens) of human cells: They are deleted from the repertoire by the induction

of apoptosis Immature B cells that bind soluble self-antigens

(monovalent self-antigens) are rendered unresponsive or anergic to the antigen As a consequence express low levels of IgM at the

cell surface and can enter the peripheral circulation where they express IgD but remain anergic

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Binding To self-Ag In The Bone Marrow Can Lead To The Deletion Or Inactivation Of Immature B Cells

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Replacement Of L-chains By Receptor Editing Can Rescue Some Self-reactive B Cells By Changing Their Ag

Specificity

When a developing B cell produces surface IgM that are strongly cross-link by multivalent self-antigens (MHC complex molecule on cell surfaces) the B cell undergoes developmental arrest The amount of IgM on the surface is reduced and

the RAG genes are not turned off Continued synthesis of RAG proteins allow the cell

to continue L-chain gene rearrangement Usually leads to a new productive rearrangement

and expression of a new L-chain which combines with the previous H-chain to form a new receptor (receptor editing)

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Replacement Of L-chains By Receptor Editing Can Rescue Some Self-reactive B Cells By Changing Their Ag

Specificity

Receptor editing If this new receptor is not self-reactive the cell is

“rescued’ and continues normal development much like a cell that had never reacted with self

If the cell remains self-reactive, it may be rescued by another cycle of rearrangement but if it continues to react strongly with self it will undergo apoptosis and be deleted from the repertoire

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Developing B cells strongly interacts with self-antigen.

Continued synthesis of RAG proteins allows light chain rearrangement.

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Architecture of the Lymph Node

• In LN, there are discrete sites for B cells and T cells.

• Effector B cells; plasma cells -secrete antibodies.

• Expansion occurs in lymphoid follicles.

• As lymphocyte development proceeds, follicle shape changes - germinal center.

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The Circulation Route Of B Cells Through A Lymph Node

From the bone marrow, an immature B cell will migrate via the blood to the secondary lymphoid organs, directed by cytokines

B cells enter the cortex of the lymph node through the wall of specialized high endothelial venules (HEV), also directed by cytokines

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Final Maturation of B-cells

B-cells interact with Follicular dendritic cells to receive their final signal to fully mature and survive.

Now called naïve B-cells (mature) Immature B-cells that fail to enter the follicle will die.

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Encounter With Antigen Leads To The Differentiation Of Activated B Cells Into Plasma Cells And Memory B Cells

Secondary lymphoid tissue are the sites where mature, naïve B cells encounter specific antigen

Antigen-specific B cells stay in the T-cell areas, and are activated by Ag-specific, CD4 helper T cells T cells provide signals that activate the B cells to proliferate and

differentiate In lymph nodes and spleen some of the activated B cells

immediately proliferate and differentiate into plasma B cells and secrete antibody (lower affinity)

Other activated B cells migrate to a primary follicle that matures into a secondary follicle containing a germinal center

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Encounter With Antigen Leads To The Differentiation Of Activated B Cells Into Plasma Cells And Memory B Cells

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B cells are found in the dark zone, light zone and mantle zone.Large proliferating B cells are the centroblasts in dark zone.Centroblasts mature into non-dividing centrocytes that have undergone

Ig class switching and somatic hypermutation.Centrocytes and Follicular dendritic cells occupy the light zone.Large masses of helper T cells are in the T cell zone and light zone.

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Selection After Affinity Maturation

Cells that survive the selection process after affinity maturation undergo further proliferation and migrate from the germinal center to other sites in the secondary lymphoid tissues or bone marrow

Selected B cells differentiate into plasma cells secreting high affinity, isotype-switched antibody

As immune response subsides, germinal center B cells develop into memory B cells capable of making high affinity antibody when re-exposed to the same antigen (basis of secondary immune response)

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Population Dynamics Of B Cells

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Population Dynamics Of B Cells

B-cell population = continually changing and heterogeneous population Different states of development and differentiation Immature B cells produced bone marrow have

Igs specific for the entire range of molecules found in biological systems

B cells with receptors that bind to self-antigen present in the bone marrow Eliminated by clonal deletion or inactivated

Passage through lymphoid follicles is essential for sustaining mature, naïve B cells in the peripheral circulation No follicle visit die Follicle visit unless stimulated by specific-Ag

die Proliferation, differentiate memory B cells

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A Summary Of The First Two Main Phases Of B-cell Development

Stages in B-cell development

Stem cell in bone marrow mature, naïve B cell Location of B cells at

different stages State of the Ig H-

and L-chain genes Form of Ig

expressed at each stage

Recall: B-2 cells

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A Summary Of The Last Two Main Phases Of B-cell Development

Stages in B-cell development from the activated mature B cell terminally differentiated plasma cell

Plasma cells can differentiate directly from activated B cells, from isotype switched, somatically hypermutated centrocytes or from memory B cells

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