Development of B cells

Question 1

B cell development can be grouped into 4 sequential steps.

What are they?

Answer 1

Stage 1: There is generation of B-cell receptors in the bone marrow.

Stage 2: Negative selection in the bone marrow.

Stage 3: Migration of B cells through the circulatory system to secondary lymphoid organs and B-cell activation through binding to foreign antigens.

Stage 4: Activated B cells give rise to plasma and memory cells. Plasma cells secrete antibodies.

Question 2

(T/F) B cells can recognize antigens directly unlike T cells where antigens are presented to them on the surface of APC cells on MHC complexes.

Answer 2

True!

Question 3

What are the three main mechanisms that drive precursor B cells to develop into mature B cells?

Answer 3

1) Signals transmitted by bone marrow stromal cells

2) Rearrangement and expression of immunoglobulin genes

3) Negative selection in the bone marrow

Question 4

1) What are bone marrow stromal cells?

2) Why are these cells important for B cell development?

Answer 4

1) Bone marrow stromal cells are non-lymphoid cells that form a network of connective tissue within the bone marrow.

2) They form specific ADHESIVE CONTACTS with the developing B cell lineage by establishing interactions between cell-adhesion molecules and their ligands.

They create a SPECIALIZED MICROENVIRONMENT within the bone marrow that delivers SIGNALS to the developing B cell that SWITCH on GENES that DIRECT the B cell developmental program (progress to next step of development).

For example, the stromal cells provide soluble and membrane bound CYTOKINES and CHEMOKINES that provide signals for B cell proliferation and differentiation.

Question 5

B cell development begins in _____ _______ and is completed in _______ lymphoid organs.

B cell migrates through distinct regions of the bone marrow as they mature. Earliest stem cells reside in __________ (inner cavity of long bones).

As the B cell lineage matures, they migrate to the ________ ______ of the marrow cavity.

The transition of an immature B cell to a mature B cell occurs in the ________ lymphoid organs.

Answer 5

Bone marrow; peripheral

Endosteum

Central sinus (where they can leave the bone marrow)

Peripheral

Question 6

(T/F) As the developing B cells move through the bone marrow, cells become more mature and rely more on stromal cells.

Answer 6

False!

As the developing B cells move through the bone marrow, cells become more mature and rely LESS AND LESS on stromal cells.

Question 7

(T/F) While an immature B cell expresses both IgM and IgD on its surface, the mature B cell expresses only IgM.

Answer 7

False!

The immature B cell expresses ONLY IgM on its surface and the mature B cell expresses BOTH IgM and IgD.

Question 8

(T/F) Developing B cells need different growth factors, adhesion molecules, etc, at different stages to promote maturation.

Answer 8

True! These are provided by stromal cells!

Question 9

What can be used as markers for B lineage cells at different stages?

Answer 9

As B cells develop, they express proteins that are characteristic of each stage. Many of these proteins are expressed on the cell surface and are useful markers for B lineage cells at different stages.

Question 10

The binding of B cell progenitors to ligands on _______ cells promotes the ______ expression of genes required for further B cell _________.

Answer 10

stromal; sequential; development

Question 11

List the five stages of B cell development in the proper order.

Answer 11

Stem cell
Pro-B
Pre-B
Immature
Mature

Question 12

(T/F) B cell development proceeds through stages defined by the sequential rearrangement and expression of the immunoglobulin (BCR) genes.

Answer 12

True!

Question 13

Which statements are true?

1) All loci (heavy, kappa and lambda light) are rearranged at the same time.

2) Rearrangement occurs in a fixed sequence.

3) The BCR heavy chain locus is rearranged first during early B cell development (D-J first and then DJ-V)

Answer 13

2&3!

For 1) Only one locus is rearranged at a time.

Question 14

Match what happens in the five stages of the development of the B cells:

1) Stem cell
2) Pro-B cell
3) Pre-B cell
4) Immature B cell
5) Mature B cell

A) Formation of complete heavy chain promotes assembly of pre-BCR which involves surrogate light chains. Pre-BCR signals to terminate heavy chain rearrangement (allelic exclusion) and initiate light chain rearrangement.

B) IgM and IgD expressed.

C) Successful light chain rearrangement results in formation of the complete IgM molecule. IgM expressed on cell surface with Ig alpha and beta which signals to terminate light chain rearrangement.

D) No rearrangement of any loci; no surface Ig.

E) D-to-J rearrangement of the heavy chain locus occurs FIRST and V-to-DJ rearrangement of the heavy chain locus occurs second.

Answer 14

Stem cell: No rearrangement of any loci; no surface Ig.

Pro-B cell: D-to-J rearrangement of the heavy chain locus occurs FIRST and V-to-DJ rearrangement of the heavy chain locus occurs second.

Pre-B cell: Formation of complete heavy chain promotes assembly of pre-BCR which involves surrogate light chains. Pre-BCR signals to terminate heavy chain rearrangement (allelic exclusion) and initiate light chain rearrangement.

Immature B cell: Successful light chain rearrangement (V-to-J) results in formation of the complete IgM molecule. IgM expressed on cell surface with Ig alpha and beta which signals to terminate light chain rearrangement.

Mature B cell: IgM and IgD expressed.

Question 15

Does the rearrangement of D-to-J and V-DJ occur on both alleles (maternal and paternal) in pro-B cells?

Answer 15

No!

Rearrangement of D-to-J occurs on both chromosomes while for V-to-DJ occurs on one chromosome at a time.

Question 16

Why does D-to-J rearrangement for the heavy chain occur at both alleles (maternal and paternal)?

Answer 16

Maximizes the chances of getting a functional receptor.

Most D gene segments can be translated in all three reading frames. Increases probability of generating functional immunoglobulin.

Later stages have higher failure rates.

Question 17

For V-to-DJ rearrangement, what happens when there is a productive or nonproductive join on the

1) first chromosome

2) second chromosome

Answer 17

V-DJ rearrangement occurs only on one chromosome at a time.

1) If there is a productive join on the first chromosome, cell progresses into the pre-B stage. If there is a nonproductive join in the first chromosome, V-DJ rearrangement occurs on the second chromosome.

2) If there is a productive join on the second chromosome, cell progresses into the pre-B stage. If there is a non-productive join on the second chromosome, the cell is lost.

*progressing into the pre-B cell = light chain gene rearrangement

Question 18

What are the three major components of the pre-B cell receptor (pre-BCR)?

Answer 18

1) Rearranged heavy chain

2) Surrogate light chains, VpreB and λ5 (invariant proteins, identical in all pre-B cells, structurally resemble the kappa and λ light chains)

3) Igα and Igβ signalling proteins (invariant signalling proteins)

Question 19

Signalling via the pre-BCR is antigen-__________.

Current models suggest that pre-BCR initiates signalling via _______ of unique _____ termini of VpreB and λ5.

Answer 19

Independent (still in the bone marrow)

Dimerization; amino

Question 20

What are the four signals transmitted by the pre-BCR?

Answer 20

1) Proliferation of pre-B cell

2) Promotes light chain rearrangement

3) Termination of surrogate light chain expression

4) Allelic exclusion of heavy chain

By signalling via the pre-BCR, the developing B cell tests if a functional heavy chain has been produced.

Question 21

Briefly answer the following questions regarding allelic exclusion:

1) What is allelic exclusion?

2) Why is allelic exclusion important?

3) How does allelic exclusion occur?

Answer 21

1) The exclusive expression of only one of two inherited alleles encoding the Ig heavy chains (also occurs at the light chain locus)

2) Successful rearrangement at both loci could result in a B cell with two receptors of DIFFERENT antigen specificities. Allelic exclusion ensures the B cell expresses a single receptor, thus maintaining clonal specificity.

3) The pre-BCR promotes allelic exclusion, in part, by temporally regulating gene expression of RAG-1 & RAG-2. These endonucleases initiate the somatic recombination process.

Question 22

(T/F) To prevent allelic exclusion from occurring, B cells inhibit expression of RAG-1 and RAG-2 for the rest of development process after formation of the pre-BCR.

Answer 22

False!

These endonucleases initiate the somatic recombination process! Without them, the light chain would not be rearranged. Thus, RAG-1 and RAG-2 are produced again in pre-B cells.

Question 23

What are the two types of exclusion that light chain rearrangement exhibits?

Answer 23

1) Isotypic exclusion - there are two light chain loci, kappa and lambda loci. ONLY ONE of these light chains is expressed by an individual B cell.

2) Allelic exclusion - like the heavy chain, only one of the two alleles is rearranged and expressed (one of the two alleles for the kappa chain or one of the two alleles for the lambda chain)

Question 24

Light chain rearrangement is first attempted at _______ locus.

Answer 24

kappa

Question 25

What happens when there is a non-productive join during:

1) rearrangement of kappa gene on first chromosome

2) rearrangement of kappa gene on second chromosome

3) rearrangement of lambda gene on first chromosome

4) rearrangement of lambda gene on second chromosome

What happens when there is a productive join on any of the steps?

Answer 25

1) non-productive join during rearrangement of kappa gene on first chromosome leads to rearrangement of kappa gene on second chromosome (allelic exclusion)

2) non-productive join during rearrangement of kappa gene on second chromosome leads to rearrangement of lambda gene on first chromosome (isotypic exclusion)

3) non-productive join during rearrangement of lambda gene on first chromosome leads to rearrangement of lambda gene on second chromosome (allelic exclusion)

4) non-productive join during rearrangement of lamda gene on second chromosome leads to cell loss!

If there is a productive join on any of the steps, the rearrangement of the second chromosome or of the second isotype is blocked.

The rearranged light chain associates with the heavy chain and IgM is expressed on the cell surface and the pre-B cell becomes an immature B cell.

Question 26

What is the negative selection of immature B cells?

Answer 26

The elimination or inactivation of autoreactive B cells ensures that the B cell population is tolerant to self-antigens.

Question 27

What is the difference between central tolerance and peripheral tolerance?

Answer 27

The antigen receptor of the IMMATURE B cell must be tested for autoreactivity (reactivity against self-antigens). This is known as the establishment of CENTRAL TOLERANCE because it occurs in the CENTRAL lymphoid organ (bone marrow).

However, B cells that leave the bone marrow are not fully mature and require additional maturation steps in the peripheral lymphoid organs. Any self-reactive B cells that escaped central tolerance may be removed from the repertoire during final maturation - this is also known as PERIPHERAL TOLERANCE.

Question 28

List the four possible fates of immature B cells during selection in the bone marrow.

Answer 28

1) Immature B cells do not encounter antigens (no self-reaction).

2) Immature B cells encounter multivalent self-antigens.

3) Immature B cells encounter soluble self-antigens.

4) Immature B cells encounter monovalent antigens.

Question 29

Match the fates of cells to their corresponding outcomes :

1) Immature B cells that do not encounter antigen (no self-reaction).

2) Immature B cells that encounter multivalent self-antigens.

3) Immature B cells that encounter soluble self-antigens.

4) Immature B cells that encounter monovalent antigens.

A) B cells undergo RECEPTOR EDITING so that the self-reactive receptor is deleted. Alternatively (if receptor editing doesn’t succeed), cells undergo clonal deletion whereby they die by apoptosis.

B) B cells do not receive a signal to die so they mature normally but are POTENTIALLY SELF-REACTIVE. antigen concentration is too low to stimulate BCR signalling so they are CLONALLY IGNORANT (not been properly exposed to a self-antigen).

C) B cells mature normally and migrate from the bone marrow to the periphery where they mature into recirculating B cells bearing both IgM & IgD.

D) Cells are rendered unresponsive (anergic), express low levels of surface IgM, migrate to the periphery and express IgD but remain inactive. If in competition with other B cells, they are rapidly lost.

Answer 29

Immature B cells that do not encounter antigen (no self-reaction): B cells mature normally and migrate from the bone marrow to the periphery where they mature into recirculating B cells bearing both IgM & IgD.

Immature B cells that encounter multivalent self-antigens (strong signal): B cells undergo RECEPTOR EDITING so that the self-reactive receptor is deleted. Alternatively (if receptor editing doesn’t succeed), cells undergo clonal deletion whereby they die by apoptosis.

Immature B cells that encounter soluble self-antigens (weaker signal): Cells are rendered unresponsive (anergic), express low levels of surface IgM, migrate to the periphery and express IgD but remain inactive. If in competition with other B cells, they are rapidly lost.

Immature B cells that encounter monovalent antigens (weakest signal): B cells do not receive a signal to die so they mature normally but are POTENTIALLY SELF-REACTIVE. antigen concentration is too low to stimulate BCR signalling so they are CLONALLY IGNORANT (not been properly exposed to a self-antigen).

Question 30

What is receptor editing?

Answer 30

When a developing B cell expresses antigen receptors that are strongly cross-linked by MULTIVALENT SELF ANTIGENS, its development is ARRESTED.

RAG enzymes may be expressed to allow the cell to undergo light-chain rearrangement. The new light chain combines with the previous heavy chain to form a NEW ANTIGEN RECEPTOR.

Question 31

What happens when the new receptor created by receptor editing is

1) not self-reactive

2) remains self-reactive

Answer 31

1) not self-reactive: the cell is RESCUED and continues normal development

2) remains self-reactive: 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.