Chapter 6- The Development of B Lymphocytes

Question 1

Where do B cells develop?

Answer 1

The bone marrow, and then they migrate to secondary lymphoid tissues. The B-cell receptor also forms here through VDJ recombination

Question 2

Repertoire assembly

Answer 2

When B cell precursors in the bone marrow acquire functional antigen receptors through immunoglobulin gene rearrangements. This generates a diverse range of B cell receptors which are then clonally expressed

Question 3

Main function of mature B cells

Answer 3

Each mature B cell makes an immunoglobulin of a single antigen specificity

Question 4

6 phases of B cell development

Answer 4

1. Repertoire assembly
2. Negative selection
3. Positive selection
4. Searching for infection- B cells recirculate between lymph, blood, and secondary lymphoid tissues
5. Finding infection
6. Attacking infection

Question 5

Function of secondary lymphoid tissues

Answer 5

Activation and clonal expression of B cells is triggered by antigens. B cells also differentiate into antibody-secreting plasma cells and and B cells here, helping to find and attack infection. This is the final 2 stages of B cell development

Question 6

Negative selection

Answer 6

Alteration, elimination, or inactivation of B cell receptors that bind to components of the human body. These cells could cause autoimmune disease if not eliminated. Negative selection occurs in the bone marrow

Question 7

Positive selection

Answer 7

Promotion of some immature B cells to become mature B cells in the secondary lymphoid tissues. The immature B cells compete for access to the limited number of follicles in the secondary lymphoid tissues

Question 8

Pro-B cell

Answer 8

An early stage of B cell development when the B cell expresses B-cell marker proteins and rearranges its heavy-chain genes. They still have some capacity for self-renewal, as they can divide to make more pro-B cells or make cells that move on to another stage of differentiation

Question 9

Hematopoietic stem cells

Answer 9

Self renewing cells that can develop into blood cells when factors in the bone marrow microenvironment drive differentiation. Blood cells are continually self renewed in the bone marrow, but mature cells exit and circulate in the blood or lymphatic system

Question 10

Stages of maturation for B cells (6)

Answer 10

1. Hematopoietic stem cell
2. Common lymphoid progenitor
3. B-cell precursor
4. Pro B-cell (early and late)
5. Pre-B cell
6. Immature B cell

Question 11

Secondary lymphoid tissues

Answer 11

The lymph nodes, spleen, Peyer’s patches, and MALT

Question 12

Primary lymphoid tissues

Answer 12

Thymus and bone marrow

Question 13

Overview of B cell development

Answer 13

1. The B-cell precursor rearranges its immunoglobulin genes, BCRs are generated in the bone marrow
2. Immature B cells that are bound to self cell-surface antigens are removed from the repertoire. This is negative selection, which also occurs in the bone marrow
3. Migration of B cells to peripheral and lymphoid organs and activation. Mature B cells bound to foreign antigens are activated
4. Activated B cells give rise to plasma cells and memory cells

Question 14

Early pro B-cell gene recombination

Answer 14

The DH gene segment joins to the JH segment

Question 15

Late pro-B cell recombination

Answer 15

The VH gene segment joins to the DJH to give VDJH

Question 16

Which factors are necessary for B cell development?

Answer 16

Stem-cell factor and IL-7. Stem cell factor is recognized by the Kit receptor on maturing B cells. IL-7 is a cytokine secreted by stromal cells that acts on late pro-B and pre-B cells

Question 17

E2A

Answer 17

Expressed by pro-B cells- it is a B cell specific transcription factor that induces expression of early B cell factor (EBF). Together, E2A and EBF induce the synthesis of all of the proteins needed for immunoglobulin genes to be rearranged- includes RAG-1 and RAG-2, as well as other enzymes

Question 18

What is the first B cell stage that is VDJ rearranged?

Answer 18

Large pre-B cells

Question 18

Large pre-B cells

Answer 18

The first B cell stage where VDJ recombination has occurred. It is also the first stage with Ig status- these cells produce mu heavy chains. Large pre-B cells proliferate, producing small pre-B cells in which rearrangement of the light chain gene occurs

Question 19

Heavy chain rearrangement of the pro-B cell

Answer 19

Pro-B cells are the earliest identifiable cells of the B cell lineage. Heavy chain rearrangement, which always precedes light chain rearrangement, occurs during this stage. Transcription of this rearranged heavy-chain gene produces mRNA that encodes the membrane-associated form of the mu chain (the heavy chain of the B cell receptor). Once the mu chain is expressed, the B cell becomes a pre-B cell. D to JH rearrangements occur on both alleles. Two successful
rearrangements is an
advantage since the likely
failure rate of later stages is
high

Question 20

Proteins needed for pro-B cell heavy chain rearrangement

Answer 20

Pro-B expresses E2A, which works with EBF to induce the synthesis of the proteins needed for gene rearrangement. E2A and EBF are part of a network of transcription factors. They induce expression of Pax-5, which is a transcription factor that induces the expression of proteins expressed only in B cells- Igα, Igβ, and CD19. Pax-5 is a master regulator of the rearrangement process. These proteins also induce the expression of RAG-1 and RAG-2

Question 21

Gene rearrangement mechanism (3)

Answer 21

1. Joining of VH gene segment
   to a DJH sequence occurs in
   late pro-B-cells- this occurs on only one chromosome. TdT is active here
2. There is a ~66% chance the first VH to DJH rearrangement is
   nonproductive. 45% of pro-B cells are lost at this stage
3. Successful cells move on to
   next step

Question 22

Nonproductive gene rearrangements

Answer 22

A gene rearrangement at an immunoglobulin or TCR locus in developing lymphocytes that doesn’t translate into a useful protein. The rearrangement mechanism is inherently inefficient because of the random addition of N and P nucleotides that occurs when cut V, D, and J gene segments are spliced together. Therefore, light chain genes are only rearranged when a productive rearrangement has been made at the heavy chain locus

Question 23

During recombination, which cells die by apoptosis?

Answer 23

Cells that make two nonproductive VH to DJH rearrangements die by apoptosis. This is a programmed, ordered process that is non-inflammatory. Apoptosis is also involved in selection processes and CD8 T cell mediated killing

Question 24

Apoptosis is characterized by

Answer 24

Blebbing of the cell membrane and fragmentation of the nuclei. Cells shrivel rather than lysing

Question 25

Testing of the pro-B cell heavy chain

Answer 25

The quality of the heavy chain must be assessed before the pro-B cell is permitted to rearrange its immunoglobulin light-chain genes. Pro-B cells synthesize VpreB and λ5 proteins, which form a surrogate light chain. Transcription of these proteins is controlled by E2A and EBF

Question 26

Pre-B cell receptor

Answer 26

Pro-B cells produce mu chains which form disulfide-bonded homodimers. If the dimers are of good quality, they assemble with VpreB, λ5, Igα, and Igβ to form the pre-B cell receptor, which resembles the BCR

Question 27

Allelic exclusion

Answer 27

When a cell expresses only one of its two copies of a gene. Signaling via the pre-BCR receptor promotes allelic exclusion by suppressing further H chain rearrangement (decreasing the expression of RAG-1 and RAG-2).

Question 29

Pre-B cell light chain rearrangement

Answer 29

A large pre-B cell with a rearranged H chain undergoes several rounds of division before light chain rearrangement occurs. The RAG genes, which were turned off in the large dividing pre-B cells, are reactivated during this stage so rearrangement can occur. Rearrangements take place at one light chain locus at a time, and the κ locus is rearranged before the λ locus. Only a single V-J joining event is needed to rearrange the light chain

Question 29

What happens if a functional light chain isn’t produced?

Answer 29

There are repeated rearrangements of unused V and J genes on the same allele. If this fails, there is initiation of rearrangements on the second chromosome

Question 30

What happens if light chain rearrangement is successful?

Answer 30

The pre-B cells progress to immature B cells. 85% of the cells that reach the pre-B cell stage will progress to becoming an immature B cell

Question 31

What happens if light chain rearrangement is unsuccessful?

Answer 31

Apoptosis

Question 32

What happens after cells progress to the immature B cell stage?

Answer 32

μ and light chains assemble in the ER to form IgM, and associate with Igα and Igβ. The functional BCR moves to the cell surface, and further rearrangement is shut down

Question 33

Function of transcription factors

Answer 33

Transcription factors ensure that genes are turned on and off at appropriate times. These include growth factors that support division and survival, genes involved in VDJ recombination, and signaling molecules

Question 34

Pax-5

Answer 34

A B cell-specific transcription factor. It opens and chromatin to allow for transcription

Question 35

How are rearrangement and transcription processes linked?

Answer 35

Once the cell is committed to the B cell lineage, the heavy chain locus is opened up, allowing transcription factors like Pax-5 to bind to promoter and enhancer elements and initiate transcription. This in turn facilitates the gene-rearrangement reactions, which juxtapose regulatory elements and increase the levels of transcription

Question 36

X-linked agammaglobulinemia symptoms

Answer 36

Recurrent bacterial infections (H. influenzae, S. pneumoniae, S.
pyogenes, S. aureus) no detectable levels of IgM and IgA, very low IgG levels, and absence of CD19+ cells in peripheral blood. WBC count and T cell proliferation is normal. Patients can be treated with IVIG

Question 37

Bruton’s tyrosine kinase (Btk)

Answer 37

A kinase that is critical for B cell development and survival of mature B cells. It phosphorylates phospholipase C, which modulates activity of downstream proteins

Question 38

X-linked agammaglobulinemia

Answer 38

Patients do not have a functional BTK gene. BTK is essential for B cell development, so these children have almost no antibodies because their B cells are stuck in the pre-B cell stage. Since the condition is X linked, it is mostly found in boys

Question 39

Mature B cells

Answer 39

Cells with low IgM and high IgD on their surface that can respond to antigen

Question 40

Self-tolerant

Answer 40

Immature B cells undergo negative selection, which prevents the maturation of self-reactive B cells. Therefore, the population of mature B cells in a healthy individual can’t respond to self antigens are are said to be self-tolerant. As many as 75% of immature B cells have some affinity for self antigens, so once they have been eliminated, the receptor repertoire of mature B cells is much different from that of immature B cells

Question 41

Negative selection

Answer 41

Prevents the emergence of mature B cells that are specific for common multivalent self antigens. Selection begins in the bone marrow, where immature B cells are exposed to a wide variety of self antigens expressed by stromal cells, hematopoietic cells, and macromolecules circulating in the blood plasma. Only immature B cells with receptors that don’t interact with any self antigens are allowed to leave the bone marrow and continue their maturation in the peripheral circulation

Question 42

What does the maturation process involve?

Answer 42

The alternative splicing of the heavy chain mRNA, so that the cell makes IgD and IgM. This is followed by a shift from an excess of IgM over IgD on the cell surface to an excess of IgD over IgM on the cell surface. Continuing maturation occurs in secondary lymphoid organs

Question 43

What happens to immature B cells with an affinity for self antigen?

Answer 43

These cells are signaled to arrest their developmental progression. They are retained in the bone marrow and given the opportunity to lose their self-reactivity for self antigen by altering their BCR

Question 44

Clonal deletion

Answer 44

The selective elimination of self-reactive receptor specificities from the B cell repertoire. Cells that have already undergone receptor editing are signaled to die by apoptosis if they are still self-reactive

Question 45

Receptor editing

Answer 45

The process of assessing the compatibility of receptors produced from successive gene rearrangements. If cells are self-reactive, they undergo additional light-chain gene rearrangements.

Question 46

BCR editing process

Answer 46

Binding to a multivalent self antigen in the bone marrow causes the immature B cell to continue its light-chain gene rearrangements through re-expression of RAG1/2 and rearrangement
machinery. Successive rearrangements occur until either a new non-self-reactive receptor is made or the B
cell exhausts its supply of light-chain V or J gene segments. If successful, the cell can go to secondary lymphoid tissue, if not, it dies by apoptosis

Question 47

Anergy

Answer 47

Self-reactive B cells that bind to monovalent self antigens are not signaled to continue light-chain rearrangement and are not signaled to die by apoptosis. They become inactivated and unresponsive to their specific antigens. This is called anergy. Anergic B cells don’t assemble a functional BCR, and IgM is retained within the cell. There are normal amounts of IgD on the cell surface, but the IgD doesn’t activate the B cell on binding to antigen

Question 48

Life span of anergic B cells

Answer 48

1-5 days, which is short compared to the mature B cell lifespan of 40 days

Question 49

Central tolerance

Answer 49

The combination of receptor editing, death by apoptosis, and anergy ensures that B cells leaving the bone marrow are tolerant of all self antigens of the bone marrow. Called central tolerance because it is induced in a primary lymphoid organ

Question 50

Peripheral tolerance

Answer 50

Tolerance induced to antigens outside the bone marrow. Receptor editing is not an option by this point because the recombination machinery has been shut down, but self-reactive cells can die by apoptosis or become anergic

Question 51

Chemokine receptors

Answer 51

Cytokine receptors have 7 transmembrane regions. They all signal by activating large GTP binding proteins. Each GTP protein is trimeric, containing alpha, beta, and gamma subunits. A chemokine binding to the receptor triggers the association of the G protein with the receptor. Bound GDP is exchanged for GTP, and the G protein dissociates into 2 separate units. These units function independently as second messengers, which activate intracellular molecules and signaling pathways

Question 52

Lymph node development

Answer 52

Lymph nodes contain separate B and T cell regions. Stromal cells secrete the CCL21 chemokine which attracts dendritic cells to the developing lymph node. The dendritic cells also secrete CCL21, but they secrete CCL19 too. These combinations of chemokines attract T and B cells into the lymph node. B cell areas are established by follicular dendritic cells. FDCs in the lymphoid organ secrete BLC (CXCL13) which attracts B cells from the T cell areas and creates a discrete B cell follicle

Question 53

Germinal center reaction

Answer 53

The formation of germinal centers starts when dendritic cells displaying antigen on their surface activate antigen specific CD4 T cells. These T cells proliferate and mature into effector cells, which are capable of activating antigen-specific B cells. Once activated, the B cell proliferates, and these B cells migrate to nearby follicles where they proliferate. Other B cells remain in the T cell area and secrete antibodies, but eventually die. Follicular B cells proliferate rapidly and undergo somatic hypermutation

Question 54

Germinal centers

Answer 54

Germinal centers are where B cells proliferate and undergo isotype switching and somatic hypermutation. They form within the B cell follicles in lymphoid organs