Chapter 6: The Development of B-Lymphocytes

What are the different stages in B-cell development?

6 broad stages

1. Repertoire assembly: Formation of diverse and clonally expressed BCR in the bone marrow

2. Negative selection: Elimination of self-reactive B-cell in the bone marrow

3. Positive selection: Promotion of immature B-cell to mature B-cell in secondary lymphoid tissues

4. Search for infection: Mature B-cells in blood, lymph, and secondary lymphoid tissue search for infection

5. Finding infection: Activation and expansion of B-cells in secondary lymphoid tissue

6. Attacking infection: Formation of plasma and memory B-cells in secondary lymphoid tissue

Negative selection in leads to what type of tolerance?

Central or peripheral tolerance

Central tolerance of B cells takes place in

Primary lymphoid tissue (Bone marrow)

Where does maturation of B-cell takes place?

Bone marrow

What type of B-cells leave the bone marrow?

Immature, naive B-cells

Where do B-cells finish their maturation into naive B-cells?

Secondary lymphoid tissues (lymph node, spleen, Peyer's patch)

Do stem cells have Ig genes in the germline configuration?

Yes

Which B-cell chain gets rearranged first?

Heavy chain

B cell development stages in the bone marrow

7 broad stages

1. Stem cell

2. Early pro-B cell

3. Late pro-B cell

4. Large pre-B cell

5. Small pre-B cell

6. Immature B-cell

7. Mature B-cell

Stem cell heavy and light chain genes and Ig status

H-chain: Germline

L-chain: Germline

Ig status: None

Early pro-B cell heavy and light chain genes and Ig status

H-chain: D-J rearrangement

L-chain: Germline

Ig status: None

Late pro-B cell heavy and light chain genes and Ig status

H-chain: V-DJ rearrangement

L-chain: Germline

Ig status: None

Large pre-B cell heavy and light chain genes and Ig status

H-chain: VDJ rearranged

L-chain: Germline

Ig status: μ heavy chain and surrogate light chain and pre-BCR on surface

Small pre-B cell heavy and light chain genes and Ig status

H-chain: VDJ rearranged

L-chain: V-J rearrangement

Ig status: μ heavy chain in ER

Immature B-cell heavy and light chain genes and Ig status

H-chain: VDJ rearranged

L-chain: V-J rearranged

Ig status: μ heavy chain with either κ or λ light chain and IgM on surface

Mature B-cell heavy and light chain genes and Ig status

H-chain: VDJ rearranged

L-chain: V-J rearranged

Ig status: IgM and IgD on surface

Pre-B cell receptors are found on

Large Pre-B cells

Pre-B cell receptor structure consists of

1. μ heavy chain

2. Surrogate light chain

3. Igα and Igβ

Surrogate light chain components

VpreB = Variable region of light chain

Lambda 5 = Constant region of light chain

Why are surrogate light chains needed in developing B cells?

To check for productive/non-productive rearrangement of heavy chain

If surrogate light chain is able to bind to heavy chain = productive rearrangement

Example of a non-productive heavy chain rearrangement?

Stop codon

Are most pre-BCR located on the surface of the cell?

No most pre-BCR are in the ER. Only a small amount are expressed on the surface

Small pre-B cells are a result of

Large pre-B cell proliferation

Do small pre-B cells have same heavy chain as large pre-B cells?

Yes, because large pre-B cells give rise to 80-100 small pre-B cells

Do small pre-B cells have the Pre-B cell receptor?

No

Small pre-B cells Ig μ heavy chain restricted to

Cytoplasm (ER)

Rearrangement of the Ig light-chain loci begins at which B-cell stage?

Small pre-B cell

Immature B cell express what Igs?

IgM

B cells develop in bone marrow till they become

Immature naïve B cell

Why can some B-cells be self-reactive?

Because gene rearrangement during somatic recombination is random

What will happen if self-reactive B-cells are not removed from B-cell repertoire?

Autoimmune disease

Central tolerance

Cells that bind to "self" cells are eliminated in bone marrow

B cell central tolerance takes place in

Bone marrow

Where do immature B cells go after leaving the bone marrow post central tolerance?

Bone marrow----->Circulation---->Secondary lymphoid tissue

Last stage of maturation of B cell takes place in

Secondary lymphoid tissue

What process leads to IgM and IgD expression on surface?

RNA splicing

____________ provide specialized environment for B cells at various stages of maturation

Bone marrow stromal cells

Two function of bone marrow stromal cells

1. Cell surface contact

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

When other cell adhesion molecules (CAMs) interact, they promote binding of Kit receptors on B cell to stem cell factors (SCF) on the stromal cell

VLA-4 is what type of adhesion molecule?

Integrin

Activation of Kit causes

B cell to proliferate

Function of IL-7

B cells at later stage of maturation (late pro-B cells and after) require interleukin-7 (IL-7) to stimulate their growth and proliferation

Also help with T cell growth

Gene rearrangement that cannot be translated into a protein

Unproductive rearrangement

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

Productive rearrangement

Productive rearrangement preserve a __________

Correct reading frame

What is the minimum number of rearrangements for B cell light chain?

4 minimum rearrangements possible

2 for κ chain (1 per homologous chromosome)

2 for λ chain (1 per homologous chromosome)

κ chain locus is located on

Chromosome 2

λ chain locus is located on

Chromosome 22

B cell heavy chain locus located on

Chromosome 14

True or false: Non productive rearrangement can result in apoptosis

True

How do B cells pick between the two homologous chromosome for heavy chain rearrangement?

In early pro-B cells D and J segments are rearranged simultaneously on both chromosomes. Whichever gets finished first is picked

Which light chain is rearranged first?

Kappa

What percent of B cells can make functional heavy chain?

About 50%

What percent of B cells can make functional antibodies?

Less than half

Majority of B cells have __________ (kappa/lambda) light chain

Kappa

Lambda light chain is usually picked if

No kappa rearrangement is productive

After an unproductive rearrangement of Vk to a Jk, a second rearrangement can be made by Vk2 or any other Vk that is on the __________ side of the first joint, with a Jk that is on the ___________ side of the first joint

5'; 3'

Expression of ____________ prevents further gene rearrangement

The products of Ig genes

Successful gene rearrangement is signaled by the appearance of

protein product of the gene at the cell surface by Igα and Igβ

What is the possible outcome if RAG-1 and 2 are shut down after a productive rearrangement in a B-cell?

Allelic exclusion will give homogenous BCRs with high-avidity binding

What is the possible outcome if RAG-1 and 2 aren't shut down after a productive rearrangement in a B-cell?

No allelic exclusion which would result in heterogenous BCRs with low-avidity binding

μ heavy chains assemble into dimer in __________ cells

Pro-B cells

In pro-B cells, μ heavy chains assemble into dimers in the ____________

Endoplasmic reticulum

Pre-BCR components

Heavy μ chain dimer, 2 surrogate light chains, and Igα and Igβ

Pre-BCR are assembled in

Endoplasmic reticulum

Events that take place at first checkpoint of rearrangement

Pre-BCRs form dimers and oligomer that signal that maturation can continue

Allelic exclusion of heavy chain locus

- Transcription/expression of RAG is turned off

- Destruction of RAG 2

- H-chain genes become inaccessible

Do pre-BCR interact with any ligands?

No

Pre-BCR is a what type of signal?

It is a positive signal that prevents apoptosis and allows B cell to continue dividing

After first checkpoint what is no longer made and what continues being made?

pre-BCR is no longer made

Heavy chain, Igα, and Igβ continue being made in the ER

Light chain rearrangement stage and steps

Starts at small pre-B cell

1. RAG genes are turned on and rearrangement of light chain starts

2. On completion, light chain is joined with heavy chain to form IgM

3. IgM associate with Igα and Igβ and tis transported to cell surface

Second checkpoint in B cell development signaled by

Presence of the BCR with Igα and Igβ tells the cell to halt further light-chain gene rearrangements (for the most part).

Which gene is never turned off during B cell maturation?

Genes for Igα and Igβ

Genes for Kit expressed are expressed at

Early stages of maturation

Genes for Il-7 receptor are expressed at

Early stages of maturation

They are needed for growth and proliferation at later stages of maturation

Which genes are turned on and then off and then on again?

Genes for RAG-1 and RAG-2

Are TdT genes turned off when RAG genes are turned off the first time?

There is no need to turn the genes for TdT off because they cannot function without RAG

Genes that cause cancer when their function or expression is perturbed

Proto-oncogenes

What leads to B-cell tumors during rearrangement?

Aberrant Ig-gene rearrangement

Usually caused by chromosomal translocation:

Ig gene join with a gene on another chromosome that regulates cellular growth

Viral genes responsible for transformation

Oncogenes

Burkitt's Lymphoma

Caused by translocation of MYC gene on chromosome 8 with variable region of either of three:

1. H-chain on chromosome 14

2. Kappa chain on chromosome 2

3. Lambda chain on chromosome 22

Function of MYC

Regulates cell division

Abnormal MYC expression is a result of

Translocation

Translocation of MYC can cause

Abnormal B cell growth (Burkitt's Lymphoma)

Another proto-oncogene for B cell and its function

BCL-2

Function: Protective against B cell apoptosis

Translocations probably occur during

The first attempt to rearrange a heavy-chain gene

What happens after translocation has occured?

Rearrangement 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

B-1 cells arise when?

Early in embryonic development

B-1 cells express which receptors

CD5 (usually seen on T cells)

Do B-1 cells come before B-2 cells?

Yes

Do B-1 cells require T-cell help? Why or why not?

No, because they use extensive cross-linking to override the need for T cells

B-1 cells are also known as?

CD5 B cells

Do B-1 cells need CD5 to function?

No

B-1 cells are part of which immunity?

Innate immunity

Do B-1 cell have memory?

No

Do B-1 cell perform somatic recombination, somatic hypermutation, or isotype switching?

They only perform somatic recombination

Do B-1 cell express IgD?

Little or no IgD on surface

B-1 cell Heavy Chain and light chain Rearrangement characteristics

1. V gene segments closest to D gene segments are used (H-chain)

2. V gene segment closest to J gene segment are used (L-chain)

3. TdT is not expressed in prenatal period, so no N nucleotide addition. As a result, less junctional diversity

B-1 cell secreted Ig characteristics (affinity and specificity)

Low affinity and polyspecificity (can bind to many different antigen)

What types of antigens are B-1 antibodies made against?

Bacterial polysaccharides and carbohydrates

True of false: B-1 cell antibodies are made against proteins?

False

Postnatal B-1 cells vs Prenatal B-1 cells

Use more diverse V segments and N nucleotides