The Development of Lymphocytes: B Cell Development in the Bone Marrow & Peripheral Lymphoid Tissue Deborah A. Lebman, Ph.D.

OBJECTIVES

1. To understand how ordered Ig gene rearrangements lead to the development of monospecific B cells.

2. To know the role of developmental regulation of genes involved in Ig rearrangement in B cell development.

3. To know how aberrant gene rearrangements contribute to B cell tumor development.

4. To know the differences between B-1 and B-2 (conventional) B cells. 5. To know the mechanisms that prevent development of self-reactive B cells. 6. To know the stages of B cell development that occur in peripheral lymphoid

tissue. 7. To know the normal counterparts of B cell tumors.

READING

Parham, The Immune System, Chapter 4, pp. 99-123.

DEFINITIONS AND ABBREVIATIONS

• Apoptosis; programmed cell death • Anergy; inability to respond • oncogene; genes involved in cell growth. When they become defective in

structure or expression, a cell can proliferate abnormally and form a tumor. • Surrogate light chain; proteins that interact with μ (mu) heavy chain to form pre-B

cell receptor • CAM; cell adhesion molecule • FDC; follicular dendritic cell • HEV; high endothelial venule • Key cytokines: • SCF; stem cell factor which is produced by bone marrow stromal cells and

interacts with kit receptor • IL-7; critical cytokine in early B cell development

Background

B cells continue to develop throughout your life. New B cells are generated in bone marrow, and clones of B cells expand and mature in the periphery in response to invaders. The life of a B cell can be divided into four broad phases:

1. generation in the bone marrow, 2. elimination of self-reactive B cells in the bone marrow, 3. activation in secondary lymphoid tissue 4. differentiation to plasma or memory cells in secondary lymphoid tissue

Part I: The Development of B Cells in the Bone Marrow

1. Contact with bone marrow stromal cells delivers signals that stimulate B cell development

Immature stem cells or lymphoid progenitors are located in the subendosteum. These cells express surface molecules called cell adhesion molecules (CAMs) as well as integrins, e.g., VLA-4. Stromal cells in the bone marrow have ligands/counter-receptors for these molecules. The ligand-receptor interaction between the stem cells and the bone marrow delivers a signal that leads to expression of other receptors on the early pro-B cells. An important receptor is kit. Stromal cells make stem cell factor (SCF) which binds to kit. The early pro-B cells make begin to make the receptor for IL-7 (IL7R) as they develop into late pro-B cells. IL-7, which is also made by stromal cells, is a critical cytokine involved in B cell development. During this time the B cells are actually moving around the bone marrow. Ultimately they reach the immature B cell stage at which point development is no longer contact dependent.

Figure 1. B cell development depends on interaction with bone marrow stromal cells. Note the key interactions: VLA-4-VCAM-1; Kit-SCF; IL7R-IL-7. The bottom panel a is a light micrograph of B cells in culture. It shows the intimate association between lymphocytes and stromal cells. The bottom panel B is an electron micrograph showing the same thing. Cell-cell interactions (a.k.a. cell contact, intimacy) are important in all stages of B cell development.

2. The stages of B cell development can be defined by the status of Ig genes

Figure 2. The status of Ig genes in the different stages of B cell development.

3. The rearrangement of Ig genes occurs in a defined order

Figure 3. The order of Ig gene rearrangements

The first rearrangement is D-J joining on Ig heavy chains. This process can occur on both H chain alleles. The second rearrangement is V to DJ. You will recall from our discussion on the development of the antibody repertoire that joining is imprecise. Consequently, it is possible that a stop codon is introduced during joining. This event would make the generation of a H chain protein impossible and is referred to as a non(un)productive rearrangement.

Rearrangements that lead to generation of a functional H chain are called productive rearrangements. The light chains rearrange afterward heavy chains, κ (kappa) rearranges before λ (lambda). Since only two gene segments are involved in light chain rearrangements, it is possible for light chains to undergo a second rearrangement to “rescue” the B cell.

Figure 4 A non-productive light chain rearrangement can be replaced by a second gene rearrangement.

4. Each gene rearrangement affects protein expression and subsequent gene rearrangements.

A key feature of B cell development is the generation of monospecific cells, i.e., cells that recognize only one antigen. As we have discussed, both H chains can undergo rearrangement. So what stops the production of two H chains? When a productive H chain rearrangement occurs, that H chain associates with another protein called surrogate light chain and gets expressed on the membrane of the pre-B cell. The resulting tetramer is called the pre-BCR. The pre-BCR associates with Igα (Ig alpha) and Igβ (Ig beta). Although it is not clear what the ligand for the pre-BCR is, it is clear that ligation of the pre-BCR leads to both the

cessation of H chain rearrangement and the initiation of light chain rearrangement. Once productive light chains are made, a complete BCR is expressed. Expression of a BCR somehow tells a B cell to stop rearranging light chains.

Figure 5. Productive rearrangements permit expression of “receptors” on the B cells. The receptors deliver signals to the B cells which induce the B cell to either rearrange or stop rearranging Ig genes.

5. B cell development depends on regulated expression of proteins involved in Ig gene rearrangement, signal transduction and transcription.

Figure 6. Proteins involved in recombination, signaling and transcription are expressed at specific stages of B cell development.

Some of the key proteins to take note of are:

RAG-1 and RAG-2 expression correlates with the timing of gene rearrangement. TdT (the enzyme that adds N nucleotides) is only present when H chains rearrange Igα and Igβ are signaling proteins that are expressed once heavy chains are rearranged.

Btk or Bruton’s tyrosine kinase is critical to B cell development. People who lack Btk have virtually no antibody. This syndrome is called X-linked agammaglobulinemia.

6. Aberrant gene rearrangements can lead to B cell tumors

The cutting and splicing of Ig genes occasionally goes awry, and Ig genes end up spliced to genes on other chromosomes. This is called translocation. In B cell tumors Ig genes become spliced to genes that control cell growth. Proto-oncogenes are genes that cause cancer when regulation of their expression is altered. Burkitt’s lymphoma is associated with translocations involving the myc proto-oncogene and either H or L chain genes. Another common translocation in B cell tumors occurs between the proto-oncogene BCL2 and Ig genes.

Figure 7 Cryptic RSS in other genes occasionally results in aberrant recombinations that juxtapose Ig genes with proto-oncogenes.

Part II: Selection and Development in the Periphery

1. Elimination of self-reactive B cells occurs by two routes

a. Immature B cells that recognize multivalent self antigens are eliminated

To become a mature B cell, an immature mIgM expressing cell needs to express mIgD (remember...this is accomplished by alternative RNA splicing) and emigrate from the bone marrow. If an immature, mIgM only B cell recognizes a self antigen that is present on the surface of a cell (generally these are multivaltent glycoproteins, proteoglycan, and glycolipids ) it will apoptose (die by programmed cell death). Since a dead cell cannot encounter antigen and expand, this process is called clonal deletion.

b. Immature B cells that recognize soluble self antigens are anergized

If a mIgM only B cell is specific for a soluble self antigen, it will survive and migrate to the periphery expressing both mIgM and mIgD. When it re-encounters its antigen, it will not respond, i.e., proliferate mature to Ig secretion. The inability to respond is called anergy.

Figure 8. B cell selection in the bone marrow. B cells that recognize self antigen are either eliminated or anergized.

2. CD5 B Cells: A second population of B cells that develops first

a. A minority subset of B cells expressing the CD5 glycoprotein arises early in embryonic development.

Since this population arises first, this subset of B cells is called B-1 The majority population, which is what we have been discussing is sometimes called B-2 B1/CD5 cells are the major B cell population in pleural and peritoneal cavities

b. CD5 B cells come from a self-renewing stem cell that first appears in fetal life

c. CD5 B cells are less diverse than conventional B cells

TdT is not present during VDJ rearrangement in early CD5 B cells. The antibodies produced by this population are both less diverse and of lower affinity. Because they are of low affinity, they can bind to multiple similar antigenic determinants. B-1 B cells that develop later in life (when TdT is present) are more diverse. Ultimately they stop being made in the bone marrow, and in adult life are maintained by division of existing cells (self-renewal)

d. CD5 B cells respond well to carbohydrates and poorly to protein antigens.

Conventional B cells are just the opposite.

e. CLL (chronic lymphocytic leukemia) is frequently a B-1 tumor

Figure 9. A comparison of B1 or CD5 and conventional or B2 B cells.

3. Activation in Secondary Lymphoid Tissue

a. B cells leave the bone marrow and recirculate

B cells move between blood, lymph and secondary lymphoid tissue, i.e., spleen, lymph nodes, and mucosal associated lymphoid tissue (MALT). They try to get into primary follicles which are organized structures containing B cells and follicular dendritic cells (FDCs).

Figure 10. Circulating B cells enter the cortex of lymph nodes through the walls of HEV (high endothelial venules). If a B cell does not encounter antigen, it passes through the primary follicle and exits via the efferent lymphatics.

Note: A large number of B cells congregate in MALT which is Peyer’s patches, appendix and tonsil. You have more lymphoid tissue in your gut than anywhere else. The competition for entry into primary follicles is fierce. If a B cell doesn’t get into a follicle, it dies. If it doesn’t see antigen, it dies.

b. B cells that find their antigen form a germinal center

If a B cell contacts its antigen when it enters lymphoid tissue, it will get momentarily trapped in the T cell zone providing that an antigen specific T cell is also present. It will form a “primary focus” or little cluster of B cells. When cells from the cluster move into the primary follicle, the primary follicle changes its morphology and becomes a secondary follicle which contains a germinal center (The antigen-activated B cells become the lymphoblasts that populate the germinal center).

Figure 11. A circulating B cell encounters its antigen. Antigen enters lymph nodes through afferent lymphatic vesicles. The B cell encounters its antigen and receives help from a CD4 T cell that recognizes the same antigen. The activated B cell can either form a germinal center or mature to a plasma cell which will exit via the efferent lymphatics

4. Differentiation to plasma cells and memory cells in secondary lymphoid tissue

a. In the germinal centers, the B lymphoblasts become plasma cells or memory cells.

b. Some plasma cells are formed without the B cell entering a follicle.

In spleen and lymph nodes, when they initially contact antigen, some of the B cells will mature to plasma cells. It isn’t necessary to enter a follicle for this to occur.

Figure 12. Distinguishing features of B cells and plasma cells.

5. B cell tumors represent different stages of B cell development

a. Tumors retain many characteristics of normal cells.

b. The location of a B cell tumor is indicative of its normal counterpart.

Follicular center cell lymphoma is derived from mature naive cells and grows in follicles Myeloma is derived from a plasma cell and grows in the bone marrow.

Figure 12. B cell tumors are derived from different stages of B cell development.