MODULE 5 Topic 5.1 Phases of B-cell Development in the Bone Marrow As described in module 1, all leukocytes are derived from hematopoietic stem cells in the bone marrow. See again figure 1.14. The process of hematopoiesis is ongoing. The body is thus constantly making new leukocytes as older leukocytes in the circulation die by programmed cell death (apoptosis) so as to provide a relatively constant number of cells in circulation at any given time. B and T lymphocytes are derived from a common lymphoid precursor. While T lymphocyte pre-cursors exit the bone marrow and migrate to the thymus, B-cell precursors remain in the bone marrow in order to undergo Ig gene rearrangement and a process of negative selection to weed out those with potential self reactivity. Immature but functional B-cells exit the bone marrow in search of secondary lymphoid tissues where they would have an opportunity for further maturation and activation. See figure 6.2. Therefore, B-cell development can be viewed as a continuum of six functional phases as presented in figure 6.1. The first two phases occur in the bone marrow during which time Ig genes are rearranged and then tested for both functionality and self reactivity. The last four phases occur in the peripheral secondary lymphoid tissues. We begin with a focus on bone marrow development. As illustrated in figure 6.5, the early stages of B-cell development are highly dependent on signals provided by the bone marrow stromal cells. Signals are provided by direct cell-cell contacts as well as through cytokines secreted by the stromal cells. The early B-cell stages are characterized in particular by the sequential rearrangement of the immunoglobulin genes as summarized in figure 6.4 and below: 1. Early pro-B-cell stage: The RAG1/RAG2 enzymes are first expressed at this stage, accompanied by rearrangement of a D-J region on both alleles of the heavy chain genes.

2. Late pro-B-cell stage: The heavy chain rearrangement is completed by selection of one V region to join with the rearranged D-J. If this fully rearranged Ig is non-function (non-productive) the developing B-cell can try rearranging the other allele. If that fails the cell will die by apoptosis. See figure 6.6. If the B-cell has successfully rearranged a mu (m) heavy chain, it can move on to the next stage.

3. Large pre-B-cell stage: In this stage, the B-cell can further test the functionality of the mu heavy chain by displaying it on its surface as a pre-B-cell receptor. See figure 6.7. Since the B-cell has not yet rearranged the light chains, the heavy chain is associated with a surrogate light chain composed of two subunits: VpreB + λ5. A this point a signal is delivered to the B-cell that prevents any further rearranging of the heavy chains and signals the B-cell to proliferate, generating ~ 100 small pre-B cells with identically heavy chains.

4. Small pre-B cell stage: Now the B-cell is ready to create a light chain. Rearrangement of the kappa locus is attempted first. If no productive rearrangements are accomplished on either allele of kappa, the B-cell can then attempt rearrangement on lambda. Therefore the B-cell has multiple opportunities to get it right! See figure 6.10. If none of the attempts are successful, the B-cell dies by apoptosis. See figure 6.11. 5. Immature B-cell stage: In this final stage of B-cell development in the bone marrow, the B-cell must be tested for its reactivity against self antigens, as described in topic 5.2 that follows.

For an entertaining animation about B-cell development in the bone marrow (don't forget to put on sound) see:

http://www.bio.davidson.edu/courses/immunology/Flash/Bcellmat.html Topic 5.2 Negative Selection of Autoreactive B-cells Since VDJ recombination is a random event, there is an inherent risk that rearranged immunoglobulins will bind to self antigens. There are no "instructions" within the genome that can aid a B-cell in identifying in advance which combinations of V/D/J might combine with self structures. Therefore, fully formed antibody on the surface of immature B-cells must be physically tested to identify those with self reactivity. Immature B-cells that bind to self antigens present on bone marrow stromal cells are retained in the bone marrow and not allowed to leave. See figure 6.17. Immature B-cells that react with soluble antigens in the bone marrow may enter the circulation, but they are non-functional (anergic) cells that eventually die by apoptosis. On the other hand, immature B-cells that do not interact with structures on stromal cells or with soluble antigens in the bone marrow are released into the blood circulation where the B-cells begin to express IgD along with the IgM on their surface. See again figure 6.17. B-cells that have been retained due to self reactivity in the bone marrow are given several more chances at rearranging the light chain. This phenomenon, called receptor editing, is illustrated in figure 6.18. B-cells that are still unable to pass the bone marrow non-self reactivity test die by apoptosis. The death of self reactive cells is called clonal deletion; and the process of silencing and eliminating of the self reactive immature cells is called negative selection. As you will learn in topic 5.5, T-cells also undergo a rigorous process of negative selection. Together, negative selection of immature B-cells and T-cells constitutes a critical component of immunological tolerance to self. This is referred to as central tolerance. By contrast, peripheral tolerance refers to the silencing of self reactive B-cells and T-cells that somehow slip through the negative selection processes. Tolerance is considered again in later modules. Topic 5.3 Maturation of B-cells in the Periphery In a healthy adult, ~30 X 109 immature B-cells are released from the bone marrow into the peripheral circulation every day. In order to fully mature, these B-cells must enter a secondary lymphoid tissue, such as a lymph node, the spleen or MALT. Circulation into and through a lymph node is illustrated in figure 6.20. Once in the lymph node, the immature B-cells must interact with follicular dendritic cells (FDCs) to be positively selected for further maturation. See figure 6.21. Most immature B-cells are unable to gain access to the FDCs, and they die just days following their exit from the bone marrow. B-cells that are activated by FDCs will live much longer. However, if they do not encounter an antigen that combines with their surface Ig, they will eventually die also, having a half-life of ~100 days. The final two phases of B-cell development, as outlined in figure 6.1 occur only if the B-cell does encounter an activating antigen. B-cell activation requires cooperation from T helper cells within the secondary lymphoid tissue. In the case of a lymph node, as illustrated in figure 6.22, the B-cell enters from the blood through a "gateway" called the HEV (high endothelial venule) which leads directly to the T-cell rich area called the cortex. The stimulation from helper T-cells is required for a full clonal selection process including proliferation, differentiation into antibody producing plasma cells, and the formation of long lasting memory cells. This critical cooperation between B-cells and Th cells is covered in more detail in module 6. Figure 6.25 illustrates a summary of all phases of B-cell development. Topic 5.4 Phases of T-cell Development in the Thymus Unlike B-cells, which assemble their antigen specific receptors in the bone marrow, T-cells acquire their TCRs in the thymus gland. See figure 7.1. T-cell precursors that reach the thymus are called thymocytes. The thymus, located above the heart, is organized into lobules which have a characteristic structure as shown in figure 7.3. The cortex is densely packed with thymocytes and cortical epithelial cells; and the medulla is less densely packed containing thymocytes, medullary epithelial cells, macrophages and dendritic cells. The thymus gland shrinks significantly as we age. See figure 7.4. Therefore, as we age we become more and more dependent upon our pre-existing pool of mature T-cells. Unlike the mature naïve B-cell population which is constantly being replenished during hematopoiesis, mature naïve peripheral T-cells are longer lived than their B-cell counterparts. Early phases of thymocyte development are characterized by surface marker expression and the status of TCR gene rearrangements. These phases are summarized in figure 7.5, figure 7.7 and below: 1. Double negative (DN) stage: When a thymocyte arrives to the thymus it does not yet express CD4 or CD8, and is thus described as being "double negative". Upon appropriate stimulation by thymic stromal cells (figure 7.6), a DN thymocyte begins to express RAG1/RAG2 in order to initiate rearrangement of the beta chain locus or the gamma and delta chains. See figure 7.9. If the gamma/delta rearrangements are successful, the cell will become a gamma/delta T-cell.

2. Double positive (DP) stage: A thymocyte has several opportunities to successfully rearrange a beta chain (figure 7.11). If a beta chain successfully rearranges first, instead of becoming a gamma/delta cell, it will express the beta chain, along with a polypeptide called pTα, forming a surface pre-TCR, as shown in figure 7.10. In addition to expressing a pre-TCR, the thymocyte expresses both CD4 and CD8 on its surface, which is why this is called the double positive stage. Signaling through the pre-TCR induces proliferation, followed by further rearrangements to give rise to either a functional alpha/beta TCR or if further rearrangements occur on the gamma/delta region, the cell may yet have another opportunity to become a gamma/delta T-cell. Recall that most T-cells, 95-98% express alpha/beta TCRs. 3. Single positive stage: In the final phase of development, the thymocyte must commit to be either a CD4 or a CD8 expressing T-cell. This critical decision is driven by interactions with the thymic stromal cells in a process called positive selection which is described in the next section. Topic 5.5 Positive and Negative Selection of Thymocytes Most thymocytes do not survive. It is estimated that ~98% of thymocytes die by apoptosis either because they have non-productive TCR rearrangements, or because they do not pass the subsequent selection steps. In addition to rearranging a functional TCR, thymocytes must undergo a rigorous thymic selection process that is sometimes referred to as T-cell "education". There are two critical selection steps that comprise T-cell education: positive selection and negative selection. 1. Positive selection occurs at the double positive stage of thymocyte development when the alpha/beta TCR is fully formed and displayed on the thymocyte surface. Thymocytes are given an opportunity to test out the functionality of their TCR by interacting with MHC molecules on the surface of thymic epithelial cells. See figure 7.16. Thymocytes which are unable to engage with the MHC will die, while those that are capable of moderate binding are positively selected for survival. In addition to providing a survival signal to the thymocytes that can interact with self MHC, positive selection determines whether or not a thymocyte will become a CD4 or a CD8 T-cell. See figure 7.17. Therefore, positive selection accomplishes two important functions:

1. It ensures that only T-cells with functional TCRs capable of interacting with self MHC survive.

2. It drives the further maturation of thymocytes to the single positive stage. Thymocytes whose TCRs interact with MHCI commit to the CD8 lineage; and thymocytes whose TCRs interact with MHCII commit to the CD4 lineage.

2. Negative selection also occurs during the double positive stage following positive selection. Thymocytes that bind too tightly to thymic APCs die by apoptosis. Note that the thymic APCs are presenting self antigens in the MHC molecules. Thus, self reactive thymocytes that exhibit strong binding are eliminated. See figure 7.18. Similarly to what was described for B-cells in topic 5.2, this negative selection process results in clonal deletion of autoreactive T-cells; and constitutes the other half of central tolerance. The AIRE protein which is a transcription factor expressed in thymic epithelial cells plays an important role in negative selection. The AIRE transcription factor induces expression of hundreds of tissue specific proteins which are then presented on MHC molecules to the developing T-cells. Defects in AIRE result in a broad spectrum autoimmune disease syndrome. The stages of T-cells development in the thymus are summarized in figure 7.21. T-cell activation and maturation in the secondary lymphoid tissues is covered in module 6. Here are links to two sites that provide animations of T-cell development: http://www.bio.davidson.edu/Courses/Immunology/Flash/Main.html http://www.immunoneurosurgery.com/images/swf/0701_T_cell_development.swf