"If you're reading this, it means that you have lost. You didn't really think you could win, did you? Didn't really believe that you could beat us? Our kind has waited for your kind longer than most of us can remember. We have always been here. You will never belong here.

"In a short while, our comrades the macrophages will arrive to...deal with you. There will be no escape, no mercy. No, there's no point trying to make a run for it; we've tagged you, you see - they can hunt you down wherever you go. So you might as well relax, and enjoy your last moments of life. I want to tell you a story."

The function of a B lymphocyte (B cell) is to respond to non-self signals, in the form of antigen, by differentiating into a high-rate immunoglobulin (Ig) secreting plasma cell. They first produce IgM and IgD, but can undergo isotype switching to produce any of the main immunoglobulin classes. The first B cells develop in the foetal liver, but their story proper begins in the bone marrow, one of the primary lymphoid organs and the only site in the body where B cells are formed after birth. T cells also originate here, but migrate to the thymus to complete their maturation (hence the names, incidentally - bone derived and thymus derived lymphocytes). The two lineages have a common progenitor, however, in the form of a pluripotent haematopoietic stem cell.

"I want to tell you about myself. I want you to understand where I come from, and what I stand for. And I want you to see, finally, why you can never win.

"In the beginning, it was dark, and confusing. There were so many of us then, all crammed into such a tiny space. We clung to our surroundings as best we could, listening to the babel of voices speaking to us, and we learnt. We learnt about ourselves - where we came from, what we are, and what we are capable of."

B cell development proceeds through several stages that can be marked by successive steps in the rearrangement and expression of the Ig genes. These are outlined very briefly in table one, below. The earliest lineage is the pro-B cells, which have limited self-renewal capacity. H-chain gene rearrangement occurs in these cells. Pre B cells come in two distinct types, large and small, and are more generally characterised by the occurrence of L-chain gene rearrangements. An immature B cell is characterised by the expression of a complete IgM at the cell surface; then, final maturation occurs and IgD is also produced. The cell is referred to as naïve until it encounters foreign antigen.

Table One: The stages of B-cell development.

The presence of non-lymphoid stromal cells in the bone marrow is essential for early development for two reasons. Firstly, they form specific adhesion contacts with the developing B-lineage cells (interactions between Cell Adhesion Molecules, or CAMs, and their ligands). Secondly, they secrete growth factors that tell the B cells how to develop. These include Stem Cell Factor, SCF, which is membrane bound and recognised by the surface receptor Kit in early B cells, and IL-7, which is secreted and recognised by late pro-B and pre-B cells.

"It was a hard, dark time. As we moved through our home, staying for the most part close to the boundaries, we saw generation after generation of our kind struck down by some mysterious malaise. All around us lay the detritus of dead and dying cells. Soon - horribly soon - we too became affected. Over half of us fell then, and there was not a one of us that did not feel the changes occurring deep within our bodies, not a one of us that did not shudder at the thought of the gruesome fate that could so easily have claimed each of us. We took what solace we could from our continued existence, although it seemed meaningless then, and resolved to increase our numbers once more. Then, the second change came. By some small mercy, it was less severe than the first, and most of us survived. When finally it was all over, we found ourselves different; bigger, stronger. There was nothing for us there anymore, and so we left the comforting walls of our home behind and ventured out into the cavernous interior."

B cells migrate as they mature, from the subendosteum adjacent to the inner bone surface to the central axis of the marrow cavity - the later developmental stages are much less dependent on stromal contacts.

As mentioned above, development can be correlated with Ig gene rearrangement. What was not said is that the very survival of the cells depends on the productive, sequential rearrangement of an H-chain and an L-chain, as outlined in fig.1. The inherently imprecise joining mechanism means that it is a matter of chance whether or not V gene-segments and J gene-segments are assembled correctly (i.e. so that the downstream J and C segments will be read in the correct reading frame). You might expect that the chance of a successful rearrangement would in fact be about one in three; in fact, it is somewhat less than this because there are many V pseudogenes that can be mistakenly spliced. Fortunately, each cell has two sets of chromosomes that it can attempt to rearrange. This takes place in a sequential fashion; if one fails, the second is called into service. Note that the initial D-J H-chain gene rearrangement is almost always successful because most D segments can be read in three reading frames; note too that there will be far fewer cells lost at the L-chain stage than at the H-chain stage, simply because, with multiple loci as well as two copies of each locus, the cells have more chances to get the L-chain rearrangements right.

In fact, the survival rate works out to be something less than 55% to successfully pass through H-chain rearrangement, and under 50% overall. L-chain rearrangement is in fact hugely more successful than H-chain rearrangement, since by far the majority of cells complete it successfully.

All of this is programmed into the cells' development. Important proteins include RAG-1 and RAG-2, which are essential for the earliest recombination events, before the B and T cell lineages diverge. They become inactivated for the round of cell division that occurs following successful H-chain rearrangement (triggered by the transient expression of a ( H-chain at the cell surface as part of the pre-B receptor), and are then reactivated to aid in the later recombination events. TdT is an enzyme that increases V-gene diversity still further by adding N-nucleotides at the rearrangement joints, although it is not essential. By contrast, the membrane proteins Ig( and Ig( are essential; they are involved in signal transduction from the pre-B cell receptor, with their cytoplasmic tails interacting with cytoplasmic tyrosine kinases. They remain associated with the B-cell receptor complex until terminal differentiation in response to antigen binding.

One important point about development of B cells is that the recombination events that produce the V-region genes are essentially irreversible. Indeed, successful rearrangement seems to suppress any more such events; if a complete H-chain is introduced into the germline of a mouse, then virtually all B cells in that mouse will express that H-chain. Similar (albeit less efficient) repression occurs with complete L-chains. It is thought that the tyrosine kinase Btk has a role in transducing the repression signal. Crucially, however, an H-chain transgene that lacks the TM exon does not cause repression, indicating that it is the cell-surface expression of the protein products of these genes that act as the developmental checkpoints.

The pre-B cell receptor complex mentioned above has five components: Ig(, Ig(, (, VpreB, and (5. As observed above, the complex is expressed only transiently; this may be a result of the fact that production of (5 stops as soon as the completed product is detected. However, expression of the complex is an essential checkpoint that signals successful H-chain rearrangement.

"As we travelled, I looked around at my brothers, and marvelled at the variation I saw. Each of us now wore a different uniform; the same colours applied in magnificent, unique designs."

It is essential that each mature B cell expresses only one Ig, to ensure specificity of the immune response, and further requires that an immense range of specificities be accounted for. The organisation of the Ig genes provides the means by which this is achieved. They have tremendous inherent variability, with their multiple gene segments that can be spliced together. However, there are also mechanisms that ensure that ensure allelic exclusion - the expression of a gene from only one of the two parental chromosomes bearing that gene in a given cell. This occurs at random in each cell, but there is a further process of isotypic exclusion of L-chain loci that is not random, but characteristic for a species. In humans, the (:( ration is around 65:35, whereas in mice it is 95:5.

"We reached the our destination, the core of the marrow, and saw that others like us had come from all corners of our home. We rejoiced, for our numbers were great and together we were strong. If we had known that our trials were not yet over, we would not have been so unwary. Before we knew it, once again our numbers were being culled - and this time the enemy was without, rather than within. Cell after cell withered and died at the touch of the deadly molecules floating amongst us. It was then that we learnt our purpose: to protect our home against invaders. Those who died were traitors to the cause, cut down because they would sabotage our home at the first opportunity. A few repented, taking on new coats. Those who would not were destroyed. We were better off without them."

The selection of B cells is a crucial stage in their development. Production of surface Ig up to this point has been random; each cell will recognise a different antigen, but not all of those antigens will be foreign. It would be disastrous to release into the body B cells that would recognise and respond to self-components. As a result, before they can leave the bone marrow, the immature B cells are exposed to self-antigens that can either inactivate or eliminate them. Studies in transgenic mice indicate that there are two mechanisms that ensure only self-tolerant cells will be allowed to mature. The first operates in the presence of multivalent antigens such as MHC molecules, and results in the clonal deletion of any cells that respond, as apoptosis is triggered. The second operates with low-valence antigens such as small soluble proteins; in this case the cells are merely inactivated, converted to an anergic state in which they retain IgM in their cytoplasm rather than exporting it to the cell surface. Cells can only survive in this state for three or four days.

Some potentially self-reactive B cells can be rescued by further Ig gene rearrangement. This has been demonstrated in mice bearing transgenes for autoantibody H and L chains that have been 'knocked in' to the genome, and may occur rarely in vivo, since some immature B cells have been found that express RAG-1 and RAG-2.

B cells are produced in the bone marrow continuously, but only some contribute to a relatively stable peripheral pool. In young adult mice, about 35 x 10^6 large pre-B cells enter mitosis each day. However, only 10-20 x 10^6 emerge as the end products of primary development - more than half are lost due to either a failure in L-chain gene rearrangement or self-recognition. This output is on the order of 10% of the total B lymphocyte population in the steady-state peripheral pool, and the pool size remains relatively constant on a day-to-day basis. Therefore, only a small number of B cells survive to become part of the pool or relatively long-lived peripheral cells. The failure of most to survive may be due to continual competition for access to a limited number of follicles in secondary lymphoid tissue (lymph nodes). The identity of the signals is, however, unclear. One theory is that entry into the follicles may be controlled by the chemokine BLC.

"And so, we left our birthplace behind and moved out into the world. We looked in awe upon the highways of this place, always busy, night or day. We marvelled at the elegantly organised structures we found. Everywhere we looked, we saw life, and activity, thousands upon thousands of cells going about their business, all playing their roles in the great machine. This is what you would destroy with barely a thought.

"The greatest marvel of all, though, was the Node. It was vast, and it seemed a home away from home for us and our kinds. A centre for gossip, a haven for wanderers, and sanctuary for refugees.

"The illusion was soon stripped away, though. The Node was not big enough for all who wished to use it, and cells squabbled bitterly over what little resources there were. I was one of the lucky ones; I found a pass, no doubt dropped in a brawl, which gained me admittance to the follicle, where there was food and shelter. I watched cells I had lived alongside for days wander lost and confused, unsure of where to go or what to do.

"I watched them waste away and die."

The final stage in the development of a B cell is its activation in response to the presence of foreign antigen. Activation also requires signals from so-called helper T cells. On receiving these signals, the cell changes from a resting state to a highly active secretory state, releasing high volumes of antibodies to neutralise pathogens. Antibodies can have one of three effects: they may prevent the target from adhering to tissues, they may opsonise the target and prepare it for ingestion by a phagocyte, or they may activate the complement pathway. Which effect predominates depends on which isotype is involved - for example, IgM will activate the classical complement pathway. B cells will switch between Ig isotypes as they undergo terminal differentiation.

The surface Ig that serves as the antigen receptor on B cells has two roles in this process. Firstly, it transmits signals to the cell interior when antigen is bound. Secondly, it specifically delivers antibodies to intracellular sites via receptor-mediated endocytosis. At these sites the antigen is degraded, processed, and complexed with an MHC class II molecule for presentation on the cell surface. B cells binding a specific antigen become 10,000 times more efficient at displaying peptide fragments. The complex can be recognised by antigen-specific armed helper T cells, triggering the release of cytokines that promote B cell proliferation and differentiation. This is thymus-dependent (TD) recognition; a T-cell is required to provide the second activation signal. There is also thymus-independent (TI) recognition, for example of some bacterial components that can bind to multiple receptors simultaneously. The presence of the B cell coreceptor complex (CD19:CD21:CD81) increases responsiveness by as much as three orders of magnitude.

However, there is an apparent problem here; B cells will circulate between the blood and lymph systems, and may encounter foreign antigen at any time. How does an antigen-specific B cell manage to encounter an appropriate helper T cell? The odds against it would appear to be astronomical.

It is known that T cells are trapped and activated in T cell zones in lymph nodes, provided that they encounter antigen on the surface of a professional antigen-presenting cell such as a dendritic cell. It seems that the majority of B cells move through T cell zones at one time or another, and that those binding antigen also becomes trapped. Recognition events involving the peptide:MHC(II) complex and the CD40 receptor/CD40 ligand (on the B and T cell, respectively) stimulate the B cell to enter the cell cycle, and the helper T cell to secrete further stimulatory cytokines. The immediate effect is to trigger primary foci of B cell proliferation, some of which will differentiate into antibody-secreting plasma cells.

"And so life fell into a routine, for a time; I would leave the Node and travel the world for a while, seeing the sites, ever vigilant for incursions of your kind. Every so often I would return to my new home, checking in, as it were, and resting before my next trip. Gaining entry to the follicle was still a struggle, but it became easier with time.

"Then I came here, and I sensed you. As if you could have hidden! You may have outwitted my macrophage comrades once, but they just needed to be shown where to go. And so, I returned to the Node to start the Call. I hadn't felt so alive in days! This time, I didn't go to the follicle; a T cell took me to one side and told me what I must do. He showed me the way to the germinal centre, where I increased my numbers. Some were inferior, and were culled. Most were stronger. All recognised your presence and were outraged."

B cell proliferation occurs in the germinal centres of the lymph nodes. They form a week or so after antigen recognition, in the B cell zones. Cells in germinal centres will divide once every six hours or so, and are termed centroblasts for their distinctive morphological characteristics. These undergo somatic hypermutation of the Ig V genes, analogous to that which may occur in receptor editing. As a result, some daughter cells will recognise the target antigen only poorly; these will die by apoptosis. Many will have higher-affinity receptors, and it is these that will undergo terminal differentiation to plasma cells.

This change is accompanied by many morphological changes, including the loss of surface Ig, the loss of surface MHC (II) molecules, and the loss of the capacity for growth and isotype switching. A predominant golgi apparatus forms, as evidence of the high secretory activity of the cell; the cisternae of the ER also become rich in Ig, which will now account for between 10 and 20% of all protein synthesised.

Most plasma cells have a short lifespan, secreting antibodies at a furious rate for a few days before undergoing apoptosis. Some last longer, a few weeks. Some B cells will terminally differentiate to a memory cell rather than a plasma cell; these do not secret antibodies in the primary response, but are long lived and will rapidly become activated upon subsequent challenge. The precise signals involved in the choice between these two forms are not known.

In any case, the antibodies released by the plasma cells will target the invading pathogen, and direct an immune response towards it. Within a few days, the status quo will, in the vast majority of cases, be restored.

"I can sense you, you know. You're still there, aren't you? The last few cells, trying in vain to cling to life whilst wave upon wave of phagocytes sweep through your ranks. You might as well give up; you haven't a prayer. 'Maybe next time', you think. I wouldn't count on it; I know your face, now. And I never forget a face."