HST 175

September 19th, 2000,

1.30 to 4 P.M.

Shiv Pillai

Recommended Reading: Abbas et al., 4th edition, Chapters 7 and 9; Janeway et al., 4th edition, Chapters 6 and 9

While the initial steps of B lymphoid commitment and development depend upon signals from stromal cells, unique B lineage specific selection events drive the developmental program after immunoglobulin gene rearrangement is initiated. Two of these events are based on the nature of the rearrangements that have occurred at the heavy chain locus. Cells in which a particular D segment reading frame can be used following D to J_H rearrangement are selected against, although this phenomenon is poorly understood, and may be restricted to the mouse. Approximately one-third of developing pre-B cells in the bone-marrow make in-frame V to DJ_H rearrangements and these cells are positively selected (positive selection I). Selection events at the pre-B stage in the bone marrow are probably distinct from those in the fetal liver as will be discussed below. In the bone-marrow pre-B receptor signaling mediates both positive selection I as well as allelic exclusion at the IgH locus. Once both heavy and light chain genes are rearranged and expressed, immature B cells may also be positively selected in response to antigen receptor mediated signals (positive selection II). If these cells express self-reactive antigen receptors, they may be subject to receptor editing) or they may be selected against by means of a deletional process. An overview of the different stages of B cell development, following the nomenclature developed by Hardy and colleagues, is provided in Figure 1.

HSCs presumably respond to external stimuli that commit them to distinct differentiation programs. These external stimuli presumably initiate signaling pathways which induce specific nuclear factors to drive the process of differentiation. A challenge in the field is to connect specific ligands, receptors, and signaling pathways, to transcription factors that drive the developmental process. A number of extracellular factors may contribute to the process of B cell commitment and some of these are considered in Figure 2.

A rearranged immunoglobulin heavy chain gene encodes two forms of heavy chain protein, a membrane form and a secreted form. mRNAs specific for these two forms are generated as a result of differential splicing and polyadenylation. It is the membrane form of the immunoglobulin heavy chain that is involved in signal transduction, as part of the pro-B receptor in pro-B cells, the pre-B receptor in pre-B cells, and the antigen receptor in B cells. Broad differences in membrane and secretory immunoglobulin heavy chains are highlighted in Figure 3

Figure 3.Membrane IgM heavy chains possess a 13 amino acid (aa) negatively charged extracellular connecting piece, a 26 amino acid membrane anchor and a short cytoplasmic tail (KVK)

The membrane and secretory forms of immunoglobulin heavy chains of all immunoglobulin isotypes are generated by alternative processsing. In Figure 4 the selective polyadenylation and alternative splicing events involved in generating these two messages are highlighted. Through most stages of B cell differentiation, RNA processing generates both forms of heavy chain message. However in plasma cells the secretory form is more efficiently generated.

It is formally possible that a yet to be identified ligand exists for the pre-B receptor. However truncated immunoglobulin heavy chains lacking ligand binding regions can participate in the generation of pre-B survival and progression signals. Given the fact that pre-antigen receptors are generated, not to respond (like most receptors) to external clues, but to monitor if the "right" kind of rearrangement has taken place within a given cell, it is not unresonable to suspect that such a receptor does not require an external ligand for signal initiation. The fully assembled receptor, because of some structural feature of the surrogate light chains, may be constitutively "on" or may respond to a ligand made by the pre-B cell itself.

Fig. 5. The pre-B receptor is made up of the membrane form of the heavy chain, surrogate light chains, and the Iga/b heterodimer. Src family kinases and Syk are associated with the receptor.

a) Is there a ligand for the pre-B receptor or is signaling constitutive and dependent primarily on assembly of the receptor?

b) What are the molecules and signaling pathways downstream of the pre-B receptor?

In spite of considerable effort over a decade, no ligand has so far been discovered for the pre-B or pre-T receptors though one might well be found in the future. The model suggesting that signaling might be constitutive is supported by experiments in which a truncated m chain has been found to drive cells through the pro-B to pre-B transition. Analagous experiments have been performed in the T lineage. Although in pre-B cells that are expressing surrogate light chains at high levels prior to light chain gene rearrangement the pre-B receptor has been described on the cell surface at low levels there is no hard evidence that it si actually expressed on the cell surface. Experiments in which the pre-T receptor has been held intracellularly have attempted to address this issue but might suffer from the problem that ER retention motifs compromise association with signaling chains. However even if surface expression is required, possibly for the receptor to be in the proximity of caveoli like DIFs in order to be in proximity to signaling molecules, it may well be that these receptors are activated in a ligand independent fashion.

From a mechanistic standpoint it is unclear whether a strong signal to a self-reactive B cell actually leads to the re-expression of Rag genes or merely prevents the cell from moving on to a stage when Rag genes are shut-off. The end-result is a cell that continues to rearrange its light chain genes. Indeed multiple light chain loci might have been generated by gene duplication and preserved during evolution primarily to facilitate the process of receptor editing. Receptor editing is primarily a light chain phenomenon. V_H gene replacement is known to occur at the heavy chain locus where a new V_H gene might recombine into an exiting rearranged gene by joining with a "cryptic" heptamer in the coding region of the previously rearranged V_H gene.

Is deletional tolerance a "physiologically" relevant phenomenon in the B lineage? At present the jury is still out on this issue - the weight of the more recent evidence favors the view that multivalent self- antigens that can access the bone marrow probably induce tolerance by provoking antigen receptor signal mediated receptor editing. Other modalities of tolerance induction, such as anergy and follicular exclusion may be relevant at least for a subset of soluble self-antigens and these will be discussed in a later section in this chapter.

For most students of the immune system positive selection of T cells during thymic development seems to make intuitive sense when one considers the need to select MHC restricted T cells. It has not been readily apparent why a positive selection phenomenon might be of relevance for recently generated B cells. Of the large number of B cells generated in the bone marrow every day (about 10⁸) cells only about 10-15% actually make it to the periphery. Most newly generated B cells have a life-span that can be measured in days but a proportion of naive B cells become long-lived cells which may recirculate in the periohery for 6-8 weeks. The possibility has been raised in some studies that a proportion of newly generated B lymphocytes might in a non-random way (based on receptor specificity) be selected to develop in the periphery and to become long lived B cells.

Immature B cells (stage E) emerge from the bone marrow beteween 2-3 days after they receive the pre-B receptor signal in stage C/C’ and emerge in the spleen as a IgMhiIgDlo population (Fraction F III, according to categories described by Hardy and colleagues). The splenic IgMhiIgDlo population contains both recently generated bone-marrow derived B cells as well as a distinguishable "older" subset of B cells known as marginal zone B cells. Recently generated Fraction III cells which emerge in the spleen are distributed initially in the T cell zone and in the red pulp. Low-level BCR signals which require Syk activity might contribute to the entry of these cells into the follicles. The newly generated Fraction III cells mature in the periphery over the next 1 to 3 days into IgM high, IgD high (Fraction II) B cells. In a few days these Fraction II cells in turn mature into IgDhigh, IgM low (Fraction I) B cells which represent the most mature peripheral B cell population. Fraction II and Fraction I cells express high levels of CD23 and acquire the ability to recirculate, migrating constantly between follicular areas in the spleen and other peripheral lymphoid organs. Indeed in just a day after bone-marrow cells enter the spleen (soon after they enter Fraction II in the follicles), they acquire the ability to recirculate and seed lymph nodes.

In secondary lymphoid organs IgDhi (Fraction II and I) B cells reside largely in lymphoid follicles which may in some unknown way contribute to their extended life span. It is now clear that in the absence of Btk signals, cells that enter Fraction I are unable to become long lived B cells and rapidly die. Btk derived signals are necessary for the generation of the long-lived pool of naive recirculating peripheral B cells. The steady-state level of Fraction I cells is markedly diminished in the absence of Btk. This fraction is also decreased in the absence of CD45 which is required for Src family kinase activation and therefore presumably for Btk activation as well. The proportion of Fraction I cells increases in the absence of CD22 (which functions as a negative regulator of the BCR). It is considered likely therefore that non-mitogenic, low-intensity signals are delivered by the BCR via the Btk pathway and that these signals are essential for the maintenance of long-lived naive B cells.

What drives positive selection? Does the B cell receptor signal "tonically" as has been suggested or is it triggered in a non-mitogenic fashion by cross-reactive self antigens? Is the "tonic" signal, merely generated to tell a B cell that its bone-marrow developmental program is complete and that it should now shut off its VDJ recombination machinery and move out to where the real action is going to be? If the signal is tonic it might be reasonable to suggest that this process is NOT positive "selection" but representsa set of maturational events. It could be that what is being selected is an appropriate fit between heavy and light chain. No conclusive answer is available at present for any of the questions raised above. However it is conceivable that in the absence of a pathogen induced challenge this process is driven by available antigen and that this selection process actually evolved for a defense related reason. It is conceivable that positive selection is generally driven by non-crosslinking self -antigens and that in a viremic or otherwise infected host non-crosslinking foreign antigens may target emerging B cells and bias the peripheral B cell repertoire towards the pathogen that requires to be targeted. This could potentially result in there being more of the right kind of b cells in the periphery to drive T-dependent responses that are likley to be of protective value.

In summary, signals from the B cell receptor are required for the maturation of naive immature B cells into recirculating follicular long-lived B cells. The most mature long-lived population is made up of IgDloIgMhi cells. This maturation process may be referred to as Positive Selection II (Figure 8) to distinguish it from Positive selection I. These signals must represent non-mitogenic trophic "tickles" rather than the full fledged stimulation associated with cross-linking antigens. While there is evidence that supports the notion of cell loss somewhere between the bone-marrow and the spleen, this is based on very indirect and potentially error-prone estimations of daily bone-marrow production. There is no hard evidence to suggest that only a proportion of the emerging B cell repertoire is selected positively or negatively at the immature B cell stage. It is likely that low-intensity BCR signals delivered via the Syk tyrosine kinase assist in follicular entry (strong BCR signals as we will discuss later may do the opposite). Signals delivered via Btk may be required for peripheral B cell maintenance and therefore for the generation of long-lived B cells.

Figure 8. Positive selection II: Immature B cells are positively selected to exit the bone marrow and enter lymphoid follicles by Syk dependent mechanisms and to mature into long-lived B cells by a Btk dependent pathway

Peripheral B cell first emerge in the spleen. As mentioned above soon after entering primary follicles (follicles which lack germinal centers) they acquire the ability to recirculate and move from one follicular site to the other. this migratory behavior enables them to be available for any cognate antigen that might emerge at a given site and to be in proximity to recirculating T cells that might provide help.The following B cell populations may be identified in the spleen.

1. Recently generated B cells which are IgM^hi IgD^lo/- CD23^- and CD21 ^lo. These newly generated naive B cells cells, sometimes referred to as virgin B cells, form a portion of Faction III, and they express high levels of CD24/HSA. These cells may be found in the red pulp of the spleen and in the T cell areas (the periarteriolar lymphoid sheath in the spleen).

2. Follicular B cells are derived from the recently generated Fraction III cells. These cells colonize lymphoid follicles, express lower levels of CD24/HSA, have the ability to recirculate, and initially have a IgM^hi IgD^hi CD23⁺ phenotype (Fraction II). They mature very rapidly into IgM^lo IgD^hi CD23⁺ cells (Fraction I). These naive B cells are variously referred to as mature B cells, or follicular B cells, or recirculating B cells.

3. Marginal Zone B cells. The marginal zone surrounds the follicular area and is populated by B cells that are IgMhi, IgD-, CD23-, and CD21hi. These cells can be distinguished from newly generated fraction III cells by virtue of their higher levels of CD21. The marginal zone also contains marginal zone macrophages, and a distinct population of marginal metallophilic macrophages. Marginal zone B cells are cells that are "ready to go" and can present antigen very efficiently, and can also differentiate into plasma cells in a matter of hours. These non-recirculating cells probably represent an unusual memory population generated in the spleen (and in lymph nodes in man), by multivalent antigens such as complement coated bacterial polysaccharides which are made available on the surface of the specialized macrophages in the area. Marginal zone B cells are largely found in the spleen, and probably account for the critical role played by the spleen (as opposed to other peripheral lymphoid sites) in responses to blood-borne carbohydrate antigens. Marginal zone B cells have been considered to represent a separate lineage.

While recently generated peripheral B cells might require non-mitogenic Syk signals to enter follicles, entry (and repeated re-entry) into follicles may also require signals delivered via follicular chemokines that serves as chemoattractants for B cells. One of these is a C-X-C chemokine known as BLC (B lymphocyte chemoattractant) or BCA-1(B cell attracting chemokine-1) which signals via a chemokine receptor that is present on mature B cells known as BLR-1 (for Burkitt’s Lymphoma Receptor-1) now reclassified as CXCR5. BLC was first described in mouse tissues and BCA-1 is the human homolog of BLC. Another B cell chemoattractant is SDF-1 which activates CXCR5 and is, as we have discussed earlier, also essential for the expansion of pro-B cells in the bone-marrow. BLR-1/CXCR5 may be more critical for migration into follicles in the spleen and Peyer’s patches (since the BLR-1 knock-out mouse fails to develop follicles at these locations) while CXCR4 may be crucial at other sites. Recirculating B cells emerge from the marginal sinus or from the central arteriole, migrate through the outer T cell zone and if they are not challenged by antigen enter the adjacent follicles.

Figure 10. m and d heavy chains are generated by RNA processing of a long transcript.

Cells expressing IgD represent mature peripheral B cells and are therefore not as prone to being tolerized as immature B cells in the bone-marrow. The presence of IgD has therefore long been considered as a hallmark of maturity but we remain in the dark as to whether the IgD molecules on the cells surface function in a distinct way from IgM. IgD knockout mice have been generated but few clues have been obtained from these mice as to the functional significance of IgD. there is no firm evidence to indicate that IgD induces signaling differently from IgM. The function of IgD remains an enigma.

B cells can be triggered by two broad categories of antigen to differentiate along two distinct pathways. Multivalent antigens, exemplified by bacterial polysaccharides, can directly trigger B cells with little or no T cell help. While this category of antigens can invoke an immune response and antibody secretion the B cell is not generally triggered to go through the processes of isotype switching and affinity maturation. As aresult the antibodies generated by a T-independent antigen tend to be mainly of the IgM clas and of relatively low affinity.

Protein antigens are internalized by B cells (by receptor mediated endocytosis) cleaved into peptides and specific peptide-MHC class II complexes are presented to cognate T cells. An activated T cell may trigger a B cell by a number of means the most critical receptors on the B cell being CD40 and the IL-4 receptor. T-dependent responses result in the formation of germinal centers (described below) in secondary lymphoid organs and it in these specialized expansions of B cells that the processes of isotype switching and somatic mutation occur.

Mature B cells may encounter cognate protein antigens (T-dependent) or their receptors may be crosslinked by multivalent T-independent antigens. Protein antigens are bound by the antigen receptor, processed and presented in an MHC class II context to helper T cells. The CD40 ligand on activated T cells delivers a growth signal to B cells via the CD40 molecule on B cells. Additional signals are provided by T cell derived cytokines. T dependent signals are delivered in the germinal centers of lymph nodes and contribute to clonal expansion, the initiation of isotype switching and somatic mutation. In patients with X-linked hyper IgM syndrome, T-B collaboration is abrogated because of an inherited absence or defect in the CD40 ligand.

Upstream of each constant region is a region of DNA known as a switch (S) region. S regions contain multiple repeats of GAGCT and TGGGG. Just upstream of each switch region is an I region promoter and an I exon. When a heavy chain isotype is targeted for class switching, a transcript is initiated from the I region 5' to the targeted constant region. The transcript reads through the I exon, the switch region and the constant region exons and is terminated and polyadenylated at the 3 ' end of the constant regions. This transcript is spliced (removing the switch region and other introns). It has been postulated that the spliced RNA might be a structural component of the switch recombinase. Very little is known at present about this recombinase.

Figure 13. An outline of the process of affinity maturation

These and related findings have led to a "Transcription coupled repair" model for somatic mutation.

Goodnow, C. C. (1996). Balancing immunity and tolerance:deleting and tuning lymphocyte repertoires. Proc.Natl.Acad.Sci. USA 93, 2264-2271.

Pillai, S. (1999). The Chosen Few? Positive Selection and the generation of Naive B lymphocytes. Immunity 10, 493-502.

Rajewsky, K. (1996). Clonal selection and learning in the antibody system. Nature 381, 751-758.

1. Describe the pre-B receptor and outline its functional roles during B cell development. Distinguish between allelic exclusion and isotypic exclusion.

2. What is receptor editing? At what stage of development is clonal deletion of B cells presumed to occur?

3. What are T-dependent and T-independent antigens?

4. What immunoglobulins are expressed on the surface of naive follicular B cells? What are the mechanisms involved in the expression of these molecules?

5. What is the major site at which marginal zone B cells are found? What do these cells do?

6. What are B-1 cells?

7. How does isotype switching occur?

8. What is the germinal center reaction. What changes might a B cell undergo in a germinal center?

9. What functions are mediated by CD40 on B cells?