| Factor | Overview |
| E2A | E2A- mice do not express RAG-1, are unable to make DHJH rearrangements and fail to express λ5. |
| EBF | Early B-cell factor (EBF) is the same as E2A. |
| BSAP | Encoded by the Pax-5 gene, knockout B cells are arrested at an early developmental stage. Various B-cell-specific genes have promoters which bind BSAP, and absence of BSAP also severely impairs midbrain development. |
| Sox-4 | Although its mechanism is unclear, it is required for B cell activation. |
Before birth, the yolk sac, fetal liver and fetal bone marrow are the major sites of B cell maturation; after birth, B cells mature in the bone marrow. Many B cells are produced, but most die after a few weeks unless they encounter their specific antigen or nestle into a supportive lymphoid organ. An immature B cell bearing IgM in its membane leaves the bone marrow and matures to express both membrane-bound IgM and IgD (mIgM and mIgD) with a single antigenic specificity.
Naive B cells (have not encountered antigen) circulate in blood ad lymph and are carried to secondary lymphoid organs (notable the spleen and lymph nodes). If a B cell’s mIgM or mIgD interacts with its antigen, then the cell activates undergoes clonal expansion. This creates a population of genetically identical B cells (which express an identical antibody) that differentiates into memory B cells and plasma B cells. Also, some B cells undergo affinity maturation, whereby the average affinity of the antibodies they produce increases. Also, many B cells undergo class switching, whereby the B cells switch from producing µ isotype antibodies (IgMs) to produce γ, α or ε isotype antibodies.
| Stage | Overview | Markers | ||
| pro-B cell | B cell maturation begins when lymphoid precursor cells differentiate into progenitor B cells (aka pro-B cells) which express a transmembrane tyrosine phosphatase called CD45R (or B220 in mice). Pro-B cells require direct contact with stromal cells to develop, and their interaction is mediated via cell adhesion molecules VLA-4 (on pro-B cells) and VCAM-1 (its ligand on stromal cells). After initial contact is made, a receptor on the pro-B cell surface called c-Kit interacts with a stromal cell surface molecule known as stem cell factor (SCF). This interaction activates the thyrosine kinase activity of c-Kit, and the pro-B cell begins proliferation.
At the pro-B cell stage, heavy chain DH-to-JH gene rearrangement occurs and then a VH-to-DHJH rearrangement. If rearrangement on one chromosome is not productive, then rearrangement on the other chromosome is allowed to occur. Once heavy-chain rearrangement completes, the cell is classified as a pre-B cell. Please note that RAG-1 and RAG-2, both necessary for heavy-chain and light-chain rearrangement, are logically expressed in pro-B cells and pre-B cells. Also, the enzyme TdT is active in pro-B cells but ceases activity early in the pre-B cell stage. |
c-Kit Ig-α/Ig-β CD19 CD24 CD43 CD45R |
||
| pre-B cell | pro-B cells proliferate and differentiate into precursor B cells (aka pre-B cells) in a microenvironment of bone marrow stromal cells. Stromal cells secrete IL-7, which binds a receptor on pre-B cells that induces maturation and down-regulates adhesion molecules so that proliferating cells can detach from the stromal cells. Although direct interaction with stromal cells is no longer necessary, IL-7 secreted by stromal cells is still necessary. In the pre-B cell, the membrane µ heavy chain associates with a surrogate light chain — surrogate light chains consist of a V-like Vpre-B sequence associated noncovalently to a C-like λ5 sequence. The membrane-bound complex of µ heavy chain and surrogate light chain associates with the membrane proteins Ig-α and Ig-β to form the pre-B cell receptor, which is critical for further pre-B cell development. The pre-B cell then undergoes multiple cell divisions, with each individual progeny cell then undergoing light-chain gene rearrangement. Once a pre-B cell undergoes a productive light-chain gene rearrangement, it is considered an immature B cell. Please remember that pre-B cells still express RAG-1 and RAG-2. | pre-BCR CD19 CD24 CD25 CD45R |
||
| Immature B cell | A pre-B cell which has undergone a productive light-chain gene rearrangement is an immature B cell. Productive light-chain rearrangement finalizes the antigen specificity of the now immature B cell, as antigenic specificity is determined by both the heavy-chain VDJ sequence and the light-chain VJ sequence. Allelic exclusion means that only one light-chain isotype is expressed on a B cell membrane at any given time. Immature B cells express membrane-bound IgM (aka mIgM) along with Ig-α and Ig-β to form the B-cell-receptor (BCR). However, this IgM-bearing immature B cell is not yet functional; interaction between the BCR and a complementary antigen induces death or anergy (unresponsiveness) instead of proliferation and differentiation. | BCR CD19 CD24 CD45R |
||
| Mature naïve B cell | When the immature B cell begins co-expressing mIgD and mIgM, it is a fully functional mature naïve B cell. A naïve B cell is one which has not yet encountered antigen. mIgD and mIgM are co-expressed due to a change in processing of heavy-chain transcripts to permit production of two mRNAs — one mRNA encoding the µ membrane-bound isotype and the other encoding the δ membrane-bound isotype. mIgD is a distinctive marker of mature naïve B cells, but is not essential for proper development nor even antigen responsiveness. | |||
| Clonal Deletion | Murine bone marrow produces ∼5×107 B cells daily, but about 90% die before getting to enter the recirculating B cell pool. Much of this loss is due to clonal deletion (aka negative selection) against immature B cells which express antibodies against self antigens. If immature B cells are treated in vitro with antibodies against mIgM, the immature B cells undergo apoptosis. It is believed that if immature B cells within the bone marrow bind self antigens, that they will undergo a similar in vivo apoptotic process.
However, even the in vitro experiment found that a few cells managed to survive — this was later found to be due to editing of light-chain genes. When some immature B cells bind a self antigen, maturation is arrested and intracellular concentrations of RAG-1 and RAG-2 skyrocket. If further light-chain DNA rearrangement leads to a BCR that is not self-reactive, then the cells survive negative selection and enter the circulatory system like regular B cells Clonal deletion removes cells reactive with any self antigens found in the bone marrow. However, a series of experiments found a still-unclear mechanism that sends circulating mature B cells into an anergic (unresponsive) state if they react with self antigens.. |
| Next Steps | Study B cell activation. |
|---|
When mature naïve B cell exit the bone marrow and begin recirculation, they are arrested in G0 and typically die within a few weeks unless they are activated by their complementary antigen. An activated B cells undergoes proliferation and differentiation into memory and plasma cells, going from G0 to G1, S phase and then mitosis (cell division). There are two kinds of antigens, with each activating B cells along a unique pathway: thymus-dependent (TD) antigens and thymus-independent (TI) antigens. TD antigens requires direct contact with TH cells (aka CD4 cells) and not just cytokines secreted by TH cells. The humoral response to TI antigens is typically weaker than the TD response, does not form memory cells and predominantly leads to IgM secretion (indicating an absence of class switching). This is due to the critical role of TH in affinity maturation, generating memory B cells and class switching to other isotypes.
The B cell response to thymus-independent antigens is split into two different pathways: the type-1 thymus-independent pathway (TI-1) is caused by lipopolysaccharide and other bacterial cell wall components; the type-2 thymus-independent pathway (TI-2) is caused by repetitive molecules such as bacterial flagellin and bacterial cell wall polysaccharides. Most TI-1 antigens are able to activate B cells regardless of their antigen specificity — they are polyclonal B cel activators or mitogens. TI-2 antigens activate B cells by binding mIg — however, cytokines secreted by TH cells are required for full B cell proliferation and for class switching from IgM to other isotypes. The table below describes the TD, TI-1 and TI-2 antigens as well as their effects on the humoral response.
| TI Antigens | |||
| Property | TD Antigens | Type 1 | Type 2 |
| Chemical Nature | Solube protein. | Bacterial cell-wall components. | Repetitious peptides and polysaccharides. |
| Polyclonal Activator | No | Yes | No |
| Immature B Cells | Inactivate | Activate | Inactivate |
| Mature B Cells | Activate | Activate | Activate |
| Isotype Switching | Yes | No | Little |
| Affinity Maturation | Yes | No | No |
| Immunologic Memory | Yes | No | No |
| Polyclonal Activation | No | Yes @ high doses | No |
Two distinct signalling events are needed to push the resting naïve B cell into the cell cycle: signal 1 followed by signal 2. TH cells are essential for activation of a B cell by thymus-dependent antigens. Binding of thymus-dependent antigens to a B cell’s mIg does not alone induce proliferation and differentiation without additional interaction with TH membrane molecules as well as appropriate cytokines. The steps are described below:
| Step | Overview |
| Antigen | Antigen cross-linking to the G0 B cell BCR generates signal 1. This leads to increased expression of Class II MHC molecules and costimulatory B7 on the B cell surface. The antigen-antibody complex is internalized by receptor-mediated endocytosis, and within ∼45 minutes the antigen is degraded into small peptides which are bound by Class II MHC molecules to form cell-membrane peptide-MHC complexes.
Because B cells are able to specifically bind and present antigens, they can perform antigen-presenting cell at antigen concentrations 102 to 105 lower than macrophages or dendritic cells. While macrophages and dendritic cells are effective at high antigen concentrations, B cells are the primary antigen-presenting cells at lower concentrations. |
| TH Activation | The TH cell recognizes the Class II peptide-MHC complex — its TCR binds the peptide-MHC complex and its CD28 binds B7. Together, these two interactions not only activate the TH cell but keep it bound to the B cell. Upon activation, the TH cell begins expressing CD154 (aka CD40L). A bound B and T cell is called a T-B conjugate. Interestingly, the Golgi apparatus and microtubular-organizing junction of the TH cell migrate toward the TCR and CD28 — when the TH cell begins cytokine secretion, this means that cytokines are secreted as close as possible to the B cell. Isotype switching begins, with different cytokines initiate transcription of different heavy chain constant region I gene promoters — for example, IL-4 activates the Iε promoter to begin transcription of IgE genes. |
| TH Signal | Interaction of CD40 (a tumor necrosis factor) and CD40L (a tumor necrosis factor receptor) provides signal 2. Signal 1 and Signal 2 together send the B cell into G1 and inducing it to express receptors for various cytokines. Binding of cytokines released by the TH cell (among them IL-2, -4 and -5) supports B cell proliferation and is critical is critical for B cell differentiation into memory or plasma cells (as well as continuing transcription of I genes for isotype switching).
Although CD40 is not a kinase, upon binding with CD40L it activates protein tyrosine kinases (PTKs) such as Lyn and Syk. Also, cross-linked CD40 activates phospholipase C and induces generation of IP3 and DAG. Lastly, cross-linked CD40 interacts with TNFR-associated factor (TRAF) proteins which eventually leads to activation of the critical transcription factor NF-κB. CD40/CD40L interaction causes rearrangement of VDJ regions to the new heavy chain constant region being transcribed; with this step complete, the B cell has undergone isotype switching and is now secreting a new antibody isotype. For example, excessive IL-4 secreted by the T cell will stimulate the promoter for Iε; upon CD40/CD40L interaction, VDJ will rearrange and join with the ε constant region gene so that all antibodies produced are now IgE. |
| Next Steps | Study the humoral response. |
|---|
Cross-linking of a membrane-bound immunoglobulin (mIg) with its complementary antigen initiates a signal transduction cascade that activates the attached B cell. Membrane-bound immunoglobulins have short cytoplasmic tails, rendering them unable to transduce activating signals on their own. However, each membrane-bound ligand-binding immunoglobulin associates with a single disulfide-linked signal-transducing heterodimer Ig-α/Ig-β to form the B cell receptor. Similarly, the pre-BCR consists of the Ig-α/Ig-β heterodimer associating with the surrogate light chain and µ heavy chains. Ig-α and Ig-β each contain a cytoplasmic tail with an 18-residue motif known as the immunoreceptor tyrosine-based activation motif (ITAM) which is also present in the T cell receptor (TCR). Also, just like the TCR, the BCR draws protein tyrosine kinases (PTKs) to its cytoplasmic tail upon cross-linking of the mIg by its complementary antigen.
Antigen-antibody crosslinking leads to phosphorylation of the tyrosines within the Ig-α and Ig-β ITAMs. This phosphorylation is performed by the receptor-associated PTKs Lyn, Blk and Fyn (similar to p56Lck activity on TCRs). This ITAM phosphorylation creates docking sites for the critical proteins Syk (also a PTK, analogous to the TCR’s ZAP-70) and B cell linker protein (BLNK). These critical proteins provide docking sites for further proteins. Once BLNK has been phosphorylated by Syk, it recruits Bruton’s tyrosine kinase (Btk) and phospholipase Cγ2 (PLCγ2) so Syk can activate Btk, and so that Btk can then phosphorylate PLCγ2. Once PLCγ2 has been phosphorylated, it activates early calcium signaling and the initiation of pathways dependent on protein kinase C (PKC). The pathways activated by the BCR include small G protein pathways (for growth), PKC-dependent pathways and NF-κB production pathways — note the similarities to T cell activation.
In addition to the BCR, there are two membrane-bound components which provide stimulation (the B cell coreceptor) or inhibition (CD22). The B cell coreceptor is a complex of three proteins: CD19, which provides a long cytoplasmic tail with docking sites; CD21 (aka CDR2), which is a receptor for C3d; and CD81. C3d is a byproduct of complement that coats antigens — while the immunoglobulin binds the antigen, CD21 cross-links with C3d. This forms a BCR-antigen-BCcoR complex, allowing CD19′s cytoplasmic tail to interact with Ig-α and Ig-β and undergo phosphorylation. CD19′s phosphorylated cytoplasmic tail then binds signaling molecules, including the protein tyrosine kinase (PTK) Lyn, and hugely amplifies the activating signal. This explains how naïve B cells with low antigen affinity are still able to respond to low concentrations of antigen.
CD22 delivers a negative signal that makes activation of B cells more difficult. Activation of B cells leads to phosphorylation of the immunoreceptor tyrosine inhibitory motif (ITIM) in the cytoplasmic tail of CD22. Tyrosine phosphatase then binds the CD22 ITIM, stripping phosphates from the ITAMs of neighboring signaling complexes. Since ITAM phosphorylation is the core of B cell activation, phosphate removal deactivates the cell. CD22 knockout mice develop autoimmune diseases as they age, illuminating the importance of negative regulation.
Antibody production by activated B cells is the core the humoral response: antibody effects, such as complement activation by IgM and certain IgGs, opsonization via F(c)Rs and pathogen/toxin neutralization by high-affinity IgG and IgA; and processes related to B cell activation, such as TH2 activation and cytokine production, germinal center formation, isotype switching, affinity maturation and memory cell production. The F(c) region of IgG binds to F(c) receptors, playing a critical role (along with receptors for complement byproducts) in clearing extracellular bacteria. Intracellular bacteria are cleared by cell-mediated immunity.
Antigens are grouped into thymus-dependent antigens and thymus-independent antigens. Activation by thymus-dependent antigens requires two signals: first, binding of the antigen itself to the B cell; second, binding of of a thymocyte to the B cell. Thymus-independent antigens, conversely, activate B cells on their own; in some cases, however, TH cytokine secretion (but not binding) is needed for maximum B cell activity.
