Question

1. At the molecular level, what drives B cell development in the bone marrow?
2. What is the role of stromal cells in B cell development in the bone marrow?
3. Describe the sequence of events in B cell development with respect to rearrangement of heavy and light chain Ig genes.
4. Why does Ig gene rearrangement often fail to produce a functional protein product? What mechanisms exist to overcome this problem?
5. What is “allelic exclusion” and what is its significance to the functioning of the immune system?
6. What mechanisms ensure that allelic exclusion occurs for Ig heavy and light chain?
7. What is the pre-BCR and how does it differ from authentic BCR? What is its function in B cell development?
8. What mechanisms ensure that self-reactive B cells do not emerge from the bone marrow? 9. How does clonal deletion differ from anergy? How does the nature of the antigen (membrane vs. soluble, multi- vs monovalent) determine the B cell fate?
10. What is receptor editing and what is the role of this process in B cell development?
11. What is the fate of immature B cells reactive to self-antigens that are present outside the bone marrow, but in locations where immature B cells circulate?
12. Describe the positive selection process for B cells? Where in the body does this occur? 13. Where in the body does the maturation of B cells from membrane IgM+ to IgM+/IgD+ occur and what causes this maturation?

Answer 1

1. At the molecular level, what drives B cell development in the bone marrow?

B cells, also known as B lymphocytes, are a type of white blood cell of the small lymphocyte subtype. B cells express B cell receptors (BCRs) on their cell membrane.^[1] BCRs allow the B cell to bind to a specific antigen, against which it will initiate an antibody response.

B cells develop from hematopoietic stem cells (HSCs) that originate from bone marrow. B cells undergo two types of the selection while developing in the bone marrow to ensure proper development. Positive selection occurs through antigen-independent signaling involving both the pre-BCR and the BCR. If these receptors do not bind to their ligand, B cells do not receive the proper signals and cease to develop. Negative selection occurs through the binding of self-antigen with the BCR; If the BCR can bind strongly to self-antigen, then the B cell undergoes one of four fates: clonal deletion, receptor editing, anergy, or ignorance (B cell ignores signal and continues development).^[6] This negative selection process leads to a state of central tolerance, in which the mature B cells don't bind with self-antigens present in the bone marrow.

2. What is the role of stromal cells in B cell development in the bone marrow?

Stromal cells produce and release IL-7 (Interleukin 7). Therefore, they play an important role in the regulation of B cell development in the bone marrow, mainly controlling the size of the B cell compartment (from commitment to the pre-B cell stage). In the bone marrow, different types of hematopoietic cells develop. It is reasonable to hypothesize that different types of stromal cells exist and organize lineage-specific microenvironments.

3. Describe the sequence of events in B cell development with respect to rearrangement of heavy and light chain Ig genes.

B cell development begins in the fetal liver and continues in the bone marrow throughout our lives. Once a B cell can express both m and L chains on its membrane, it is officially a B cell. However, it is still immature and can be easily killed by contact with self antigen until it also expressed membrane IgD. The mature B cell that moves into the periphery can be activated by antigen and become an antibody-secreting plasma cell or a memory B cell which will respond more quickly to a second exposure to antigen. B cells which fail to successfully complete B cell development undergo apoptosis.

Lymphoid progenitor cells receive signals from bone marrow stromal cells to begin B cell development. Cytokines induce TdT and recombinase (RAG-1 and RAG-2) synthesis in CD34⁺ lymphoid progenitors. The cells undergo D-J joining on the H chain chromosome to become early pro-B cells and also begin expressing CD45 (B220) and Class II MHC. Joining of a V segment to the D-J_H completes the late pro-B cell stage.

Pro-B cells become pre-B cells when they express membrane m chains with surrogate light chains in the pre-B receptor. Surrogate L chains resemble actual L chains but are the same on every pre-B cell. Signal transduction molecules IgaIgb are also part of the pre-B receptor complex. The cytoplasmic tails of Ig heavy chains are too short to enter the cytoplasm and transmit an antigen-binding signal; Ig a Ig b signal transduction molecules have ITAMs (Immunoreceptor Tyrosine Activation Motifs) which become phosphorylated in response to antigen-BCR binding. The phosphorylation initiates a cytoplasmic signaling cascade. The cell halts recombination of H chain and proliferates into a clone of B cells all producing the same m chain. Since dividing cells are larger than resting cells, this stage is called the large pre-B cell.

5. What is “allelic exclusion” and what is its significance to the functioning of the immune system?
Allelic exclusion is a random process that occurs independently in different cells of the immune system. Allelic exclusion is a process by which only one allele of a gene is expressed while the other allele is silenced.

The allelic exclusion control ensures that each B cell expresses only one type of binding site because an antibody binding site is determined by the combination of a particular heavy and light chain. A B cell with a defined binding specificity can be efficiently selected and activated by the corresponding antigen. If expression of the Ig alleles was not regulated each B cell would express several combinations of the two different heavy and light chains and thus express a variety of antigen specificities Such unregulated B cells would not be able to mediate a specific immune response.

6. What mechanisms ensure that allelic exclusion occurs for Ig heavy and light chain?

Allelic exclusion has been observed most often in genes for cell surface receptors and has been extensively studied in immune cells such as B lymphocytes. In B lymphocytes, successful heavy chain gene rearrangement of the genetic material from one chromosome results in the shutting down of rearrangement of genetic material from the second chromosome. If no successful rearrangement occurs, rearrangement of genetic material on the second chromosome takes place. If no successful rearrangement occurs on either chromosome, the cell dies.

As a result of allelic exclusion, all the antigen receptors on an individual lymphocyte will have the same amino acid sequence in the variable domain of the heavy chain protein. As the specificity of the antigen receptor is modulated by the variable domain of the light chain encoded by one of the immunoglobulin light chain loci, the specificities of B cells containing the same heavy chain recombination event can differ according to their light chain recombination event.

7. What is the pre-BCR and how does it differ from authentic BCR? What is its function in B cell development?

During early stages of development, precursor B lymphocytes express a characteristic type of antigen receptor known as the pre-B-cell receptor (pre-BCR). This receptor differs from authentic BCRs in that it possesses a germ line-encoded surrogate light chain (SLC), which is associated with the signal transduction machinery via heavy chain (HC) proteins that have been generated by productive rearrangement of the immunoglobulin HC genes. The pre-BCR marks a key step of B-cell commitment, as it activates the B-cell-specific signaling cascade and mediates the selection, expansion, and differentiation of cells expressing a productively rearranged HC protein. Another difference between the pre-BCR and authentic BCR might be the initial event that triggers receptor activation, as the pre-BCR is activated in the absence of external ligands, while conventional BCRs require antigen for activation.

The process of B cell development in the bone marrow occurs by the stepwise rearrangements of the V, D, and J segments of the Ig H and L chain gene loci. During early B cell genesis, productive Ig H chain gene rearrangement leads to assembly of the pre-B cell receptor (pre-BCR), which acts as an important checkpoint at the pro-B/preB transitional stage. The pre-BCR, transiently expressed by developing precursor B cells, comprises the Ig muH chain, surrogate light (SL) chains VpreB and lambda5, as well as the signal-transducing heterodimer Ig alpha/Ig beta. Signaling through the pre-BCR regulates allelic exclusion at the Ig H locus, stimulates cell proliferation, and induces differentiation to small post-mitotic pre-B cells that further undergo the rearrangement of the Ig L chain genes.

8. What mechanisms ensure that self-reactive B cells do not emerge from the bone marrow?

Large numbers of B cells are produced in the bone marrow throughout life, but very few become mature B cells. Most B-cell death is the result of a normal developmental process which removes the excess of immature B cells that enter secondary lymphoid organs, such as the spleen and lymph nodes. Within the pool of immature B cells, there will be some that bear self-reactive antigen receptors, which can bind to molecules found in healthy body tissues. There is now considerable evidence to show that various specific mechanisms actively remove autoreactive B cells from the repertoire, before they have a chance to mature into cells that secrete autoantibodies.

9. How does clonal deletion differ from anergy? How does the nature of the antigen (membrane vs. soluble, multi- vs monovalent) determine the B cell fate?

Clonal deletion is the removal through apoptosis of B cells and T cells that have expressed receptors for self before developing into fully immunocompetent lymphocytes. This prevents recognition and destruction of self host cells, making it a type of negative selection or central tolerance. Central tolerance prevents B and T lymphocytes from reacting to self. Thus, clonal deletion can help protect individuals against autoimmunity.

Anergy is a term in immunobiology that describes a lack of reaction by the body's defense mechanisms to foreign substances, and consists of a direct induction of peripheral lymphocyte tolerance. An individual in a state of anergy often indicates that the immune system is unable to mount a normal immune response against a specific antigen, usually a self-antigen. Lymphocytes are said to be anergic when they fail to respond to their specific antigen.