B Cell Development

This module will help you

• see how B cell development results in B cell diversity and allotypic exclusion.
• understand what regulates the development of B cell specificity.
• learn about different functional classes of B cells and their distribution in the body.
• test your knowledge and immunology problem-solving skills.

Generation of B cells
Regulation of B Cell Development
Positive Selection of B Cells
Negative Selection of B Cells
B Cell Heterogeneity

Generation of B Cells

The immune system is remarkable for its ability to respond to a great many antigens, including newly synthesized compounds which did not exist until recently. Unusual properties of antibody diversity include the presence of variable and constant regions on the same polypeptide chain and identical V regions used with different C regions. Somatic recombination for generating antibody and TCR diversity is unique among mammalian genes. Successful synthesis of both H and L chains and their expression on the membrane are necessary for the development of B cells and mark the stages in that development.

B cell development begins in the fetal liver and continues in the bone marrow throughout our lives. The table below illustrates the stages of B cell development. Once a B cell can express both m and L chains on its membrane, it is officially a B cell. However, it is still immature and can be easily killed by contact with self antigen until it also expressed membrane IgD. The mature B cell that moves into the periphery can be activated by antigen and become an antibody-secreting plasma cell or a memory B cell which will respond more quickly to a second exposure to antigen. B cells which fail to successfully complete B cell development undergo apoptosis (programmed cell death).

Lymphoid progenitor cells receive signals from bone marrow stromal cells to begin B cell development. Cytokines induce TdT and recombinase (RAG-1 and RAG-2) synthesis in CD34⁺ lymphoid progenitors. The cells undergo D-J joining on the H chain chromosome to become early pro-B cells and also begin expressing CD45 (B220) and Class II MHC. Joining of a V segment to the D-J_H completes the late pro-B cell stage.

Pro-B cells become pre-B cells when they express membrane m chains with surrogate light chains in the pre-B receptor. Surrogate L chains resemble actual L chains but are the same on every pre-B cell. Signal transduction molecules IgaIgb are also part of the pre-B receptor complex. The cytoplasmic tails of Ig heavy chains are too short to enter the cytoplasm and transmit an antigen-binding signal; Ig a Ig b signal transduction molecules have ITAMs (Immunoreceptor Tyrosine Activation Motifs) which become phosphorylated in response to antigen-BCR binding. The phosphorylation initiates a cytoplasmic signaling cascade.  The cell halts recombination of H chain and proliferates into a clone of B cells all producing the same m chain. Since dividing cells are larger than resting cells, this stage is called the large pre-B cell.

Stages  in  B   Cell  Development
	stem cell	early pro-B cell	late pro-B cell	large pre-B cell	small pre-B cell	immature B cell	mature B cell
H chain genes	germline	D-J joining	V-DJ joining	VDJ rearranged	VDJ rearranged	VDJ rearranged	VDJ rearranged
L chain genes	germline	germline	germline	germline	V-J joining	VJ rearranged	VJ rearranged
Surface Ig	none	none	none	m chain in pre-B receptor	m chain in cytoplasm and on surface	membrane IgM	membrane IgM and IgD
RAG, TdT expression	no	yes	yes	no	yes	yes	no
Surrogate L chain expression	no	yes	yes	yes	no	no	no
Ig ab expression	no	yes	yes	yes	yes	yes	yes
btk*	no	little	yes	yes	yes	yes	yes
Membrane markers	CD34	CD34 CD45 (B220) Class II	CD45R Class II CD19 CD40	CD45R Class II pre-B-R CD19 CD40	CD45R Class II pre-B-R CD19 CD40	CD45R Class II IgM CD19 CD40	CD45R Class II IgM IgD CD19 CD21 CD40

* Bruton's tyrosine kinase

Following proliferation, small pre-B cells (no longer dividing) undergo V-J joining on one L chain chromosome. Once L chain has been successfully synthesized, it is expressed with m chain on the cell membrane and the cell is called an immature B cell. Immature B cells are very sensitive to antigen binding, so if they bind self antigen in the bone marrow they die. B cells that do not bind self antigen express d chain and membrane IgD with their IgM about the time they leave the marrow and become mature naive (resting) B cells.

Regulation of B Cell Development

Progenitor cells receive signals from bone marrow stromal cells via cell-cell contacts and secreted signals. This bone marrow microenvironment is responsible for B cell development. One set of CAMs involved in both B and T cell development is SCF (stem cell factor) on the stromal cell membrane and kit (CD117) on the lymphocyte membrane. A secreted cytokine important for both B and T cell development is IL-7, secreted by the stromal cell and bound to IL-7R on the developing lymphocyte. Signals from these binding events initiate cytoplasmic cascades resulting in altered expression of proteins required for development. As the B cells develop in the marrow, they migrate from the outer part of the marrow towards the core.

Somatic recombination in the developing B cell can be productive (result in synthesis of a functional H or L chain) or nonproductive due to introduction of a stop codon because of frame shift mutations (see Antibody Genes). Failure to make productive rearrangements and express Ig at the appropriate times during development results in the death of the developing cell. B cells have two opportunities to productively rearrange H chain (maternal and paternal chromosomes) and four opportunities to productively rearrange L chains (paternal and maternal k and l loci). Human B cells usually rearrange D_H and J_H segments on both chromosomes simultaneously. D_H can also be read in any reading frame, so all D-J rearrangements are productive. Estimates are that only about half of developing B cells make productive H chain rearrangements. These successful pre-B cells divide to make clones of B cells (the large pre-B cell stage) that can proceed to L chain recombination.

During the small pre-B cell stage, light chain V-J joining usually occurs first for k chain. If rearrangement is productive, k chain is made and the cell becomes an immature B cell expressing membrane IgM(k) BCR. B cells are able to repeat V-J joining several times if the first attempts are nonproductive; this process is called light chain rescue. If k genes are not successfully rearranged on either chromosome, l genes are rearranged. Success leads to production of IgM(l) BCR. If neither k nor l is productively rearranged, the cell undergoes apoptosis in the bone marrow. Only a minority of human pre-B cells fail to become mature B cells.

Genes encoding proteins required for somatic recombination and receptor expression are turned on and off at set times during B cell development. RAG-1, RAG-2, and TdT are expressed only during the times somatic recombination is occurring: early and late pro-B cell and small pre-B cell stages. TdT is often turned off sooner than the recombinases, so that N nucleotide additions to gene segment join in L chains are not as common as in H chain sequences. Chains for the surrogate L chain and for Ig a and Ig b proteins must be expressed for the pre-B receptor to appear on the cell membrane. Signal transduction molecules must be expressed during crucial times; a human B cell immunodeficiency, Bruton's X-linked agammaglobulinemia, results from failure to express btk.

Control of gene expression depends on soluble transcription factors which bind control regions in the DNA . Promoters are regions of DNA which bind RNA polymerase to initiate mRNA synthesis. Enhancers are other non-coding (intron) regions of the DNA which improve the function of promoters. Splicing of gene segments with looping out of intervening DNA moves promoters and enhancers closer together, stimulating mRNA synthesis. Tissue-specific enhancers are also required; for example, RAG-1 and RAG-2 recombine only Ig gene segments in developing B cells and only TCR gene segments in developing T cells.

Positive Selection of B Cells

Both B and T cells undergo positive and negative selection in the primary lymphoid organs. Positive selection requires signaling through the antigen receptor for the cell to survive. Developing B cells are positively selected when the pre-B receptor binds its ligand. (Developing T cells are positively selected for their ability to bind MHC as well as peptide.) Negative selection means that binding to the receptor results in cell death. Both immature B and T cells are negatively selected if they bind self antigen.

Signaling for B cell survival and movement through the appropriate stages of gene expression occurs through membrane pre-B receptor and membrane IgM expression. Two kinds of experiments have provided evidence to support this statement. Rearranged H and L chains can be inserted into fertilized mouse eggs to produce transgenic mice. Mice transgenic for both recombined Ig H and L chains generally do not recombine any other genes for Ig; they express the transgene H and L chains on all their B cells. Transgenic mice for H chain still recombine their L chain genes and vice versa. Therefore, presence of a rearranged V_H or V_L gene signals the B cell to suppress further recombination of that gene.

Mice in which functional genes (or parts of genes required for their functioning) have been deleted are called knock-out mice. Experiments that demonstrated the importance of membrane expression of the BCR complex for delivering these signals involved making knock-out mice for the H chain transmembrane exon (so that H chain would not be inserted into the membrane), genes for Iga or Igb (or just their ITAMs), or genes for the surrogate light chains l5 and VpreB. Eliminating any one of these proteins blocks development of B cells even if all other proteins can be synthesized or the complete pre-B receptor can be expressed on the membrane with the IgaIgb lacking ITAMs. Surrogate light chain l5 resembles the constant region of l chain but is encoded by a different gene. l5 associates non-covalently with a VpreB that resembles an Ig V domain. Since pre-B cells express many different V_H regions, it is hypothesized that the VpreB common to all pre-B cells binds a ligand which signals (through the signal transduction molecule IgaIgb ) the pre-B cell to divide and then begin light chain recombination. Similar signaling through other unidentified ligands shut off recombination

Somatic recombination leads to allelic exclusion for both H and L chains in individual B cells, since each B cell productively recombines only one H chain and one L chain gene. In a heterozygote each allele (allotype) is represented on about half of B cells and half of serum Ig molecules. Light chains also show isotypic exclusion, since an individual cell or molecule has only k or l chains. k and l are not represented equally on B cells or serum Igs. In humans k is favored over l by 65% to 35%. In mice, serum Ig is 95% k , while in cats it is 95% l. The ratio of k to l reflects the relative numbers of V region segments in each isotype and the relative efficiency of their recombination into functional L chain genes.

Once the B cell leaves the marrow, its survival appears to depend on further signals thought to be delivered in the lymphoid follicles of secondary lymphoid tissue. Competition between newly created B cells and older B cells for these signals probably maintains B cell homeostasis. For example, survival of injected transgenic B cells (whose unique receptor can be identified by flow cytometry) has been shown to depend on depletion of the host's normal B cells by irradiation.

Negative Selection of B Cells

B cells which express only IgM are killed or inactivated (negatively selected) when they bind multivalent ligands, unlike mature B cells which are activated by cross-linking of their BCR. Binding to multivalent (cell-associated) self in the marrow leads to B cell apoptosis and clonal deletion. Binding to soluble self does not kill the B cell; the cell can move to the periphery and express IgD but little IgM. These cells are anergic; they cannot respond to antigen and have a short life span. Cells which do not bind self express normal levels of IgM and IgD; if they successfully enter the lymphoid follicles, they can survive for a few weeks until they either encounter their specific antigen or die.

Although many self-specific B cells undergo clonal deletion, some can undergo further somatic recombination to make new V_H and V_L combinations that are not self-specific. The ability of receptor editing to rescue some self-specific B cells by changing their specificity has been demonstrated with mice carrying Ig transgenes encoding self-MHC-specific BCR. The few B cells which are produced in these mice are not self-specific because they have been able to make new (non-transgenic) recombinations. Both light and heavy chain V regions can be replaced during receptor editing.

In many animal species, germline diversity of Ig is nonexistent or very low. Only one or a small number of functional V, D, and J segments are available for recombination, so that all immature B cells have the same antigen specificity and bind self antigen. Immature B cell binding to self signals the cells to divide; and during division, DNA crossing over events with adjacent pseudogenes (gene segments containing stop codons) results in alterations to the V region sequences. This process of gene conversion produces diverse Ig V regions. Once cells no longer bind self, they mature and go to the periphery.

B Cell Heterogeneity

During fetal life, bone marrow stem cells give rise to a B cell with different properties than the conventional B cell; it is called the B-1 B cell. B-1 Cells have membrane CD5. They are self-renewing, meaning they can produce more mature naive cells like themselves by division in the peripheral lymphoid tissues. Conventional B-2 cells can only divide in response to antigen and give rise to memory or plasma cells in the periphery; more naive B-2 cells must be produced from progenitors in the marrow. B-1 BCR is much less diverse than that of B-2 cells. B-1 BCR is produced preferentially from only some Ig gene segments, does not have additional N nucleotides at the joins between segments, and is specific for mainly common bacterial carbohydrate antigens. B-1 cells secrete predominantly IgM and undergo very little somatic hypermutation. Because they respond to antigens found on multiple pathogens and bind many antigens with low affinity, B-1 cells and their secreted antibodies are called polyreactive. Much of the IgM found in unimmunized mice is produced by B-1 cells. B-1 cells produced after birth have more diverse Ig than those produced during fetal life, but not as diverse as that on B-2 cells. Eventually, bone marrow stem cells stop producing B-1 cells. An analogous T cell type produced early in development is the gamma/delta T cell.

B cells change their location with their stages of maturation, each location providing the microenvironment suitable for the B cell at that life stage. Stem cells produce lymphoid progenitors and pro-B cells in the marrow just under the bone. Developing B cells move toward the center of the marrow as they mature. Mature naive B cells leave the marrow and use selectins to bind addressins on blood vessel endothelium to enter peripheral lymphoid tissues, passing through T cell areas and entering the B cell areas (follicles). Peyer's patches, tonsils and appendix are predominantly composed of large follicles. The microenvironment in the MALT follicles (including the T cell cytokines made there) signals the B cells to produce IgA, while that in the lymph nodes and spleen signals the B cells to make IgG.

B cells encountering antigen and receiving appropriate T cell help in the T cell areas form germinal centers in the follicles, where they divide rapidly, and undergo somatic hypermutation and selection for B cells with higher affinity receptors. Antibody-secreting plasma cells, short-lived ones that have not passed through the follicles and longer-lived ones that have undergone hypermutation and class switching in the follicles, are found primarily in the medullary cords of the lymph nodes, the red pulp of the spleen, and in the bone marrow (primarily IgG-secreting plasma cells) or mucosal lamina propria (IgA-secreting plasma cells). Memory B cells are found predominantly in the marginal zone of the spleen, the sub-capsular sinus of the lymph nodes, and under the intestinal epithelium in the Peyer's patches and crypt epithelium of the tonsils; a few are also found in the blood.

B cell tumors arise from different maturational stages of normal B cells, and these naturally-occurring tumors have helped immunologists understand B cell development. Each tumor type has its characteristic Ig gene recombination state and homing properties. In nearly every case these tumors are monoclonal, arising from a single B cell which became a cancer cell. Monoclonality allows physicians to identify the tumor cells and track their responses to treatment.

DNA translocations resulting in activation of oncogenes are found in some B cell tumors. A translocation is the movement of a chromosome segment to another chromosome. Oncogenes are genes usually associated with regulated cell division; when their function is disrupted by translocation, unregulated growth can result. Epstein Barr Virus (EBV), which usually causes a mild childhood disease or a more debilitating infectious mononucleosis in young adults in the United States, is associated with a B cell cancer called Burkitt's lymphoma in Africa. In Burkitt's lymphoma cells, the oncogene myc is translocated under the control of an H or L chain promoter. Because these promoters are active in B cells, unregulated growth can occur in a B cell with this translocation plus other mutations. There seems to be a link between the occurrence of Burkitt's lymphoma and malaria. Another gene activated by translocation to Ig loci is bcl-2. Bcl-2 protein protects B-lineage cells from programmed cell death, so cells carrying translocated bcl-2 survive beyond their normal life span and may become cancerous.

Practice Quiz

Pick the one best answer to each question by clicking on the letter of the correct choice.

1. B cell differentiation begins with the expression of

a. membrane m chain + surrogate L chain.
b. membrane IgD.
c. Membrane IgM.
d. germline IgM.
e. RAG-1, RAG-2 and TdT.

2. Bone marrow stromal cells

a. are important because they provide physical support for B cells (hence, their name from the Greek word for mattress).
b. are present only in the center of the marrow.
c. present foreign antigen to B cells to stimulate somatic hypermutation .
d. present self antigen on self MHC to B cells for negative selection.
e. secrete cytokines such as IL-7 that signal developing B cells to divide and differentiate.

3. Cell adhesion molecules (CAMs)

a. are found only on bone marrow stromal cells.
b. are specific receptors for cytokines that promote cell-cell binding.
c. function primarily to hold developing B cells in one location until they are fully developed.
d. signal developing B cells to divide and differentiate.
e. signal developing B cells to die because they have bound self.

4. The developmental step that commits a cell to the B lineage is

a. expression of both membrane IgM and IgD.
b. expression of membrane m chain.
c. expression of recombinase enzymes.
d. joining of a V_H gene segment to a D_H gene segment.
e. joining of a D_H gene segment to a J_H gene segment

5. Which statement about B cell development is FALSE?

a. Cells which fail to synthesize and express m chains usually die.
b. Each DNA joining event in Ig genes has a 67% probability of resulting in a nonproductive rearrangement.
c. The earliest developing B cell which could be stained with FITC-anti-m chain would be a pro B cell.
d. The earliest developing B cell which could be stained with FITC-anti- k chain would be an immature B cell.
e. The enzyme which can add nucleotides not encoded in the DNA to Ig genes during recombination is TdT.

6. Once H chain genes have been productively rearranged and expressed on the pre-B cell membrane, the next event to occur in the cell is

a. death of cells binding self antigen.
b. expression of membrane IgD.
c. expression of membrane of IgM.
d. proliferation of the pre-B cells.
e. somatic recombination of light chain genes.

7. In a productive rearrangement of Ig DNA, what is produced must be

a. a functional membrane Ig protein chain.
b. a loop of DNA that is then removed.
c. an mRNA for H or L chain.
d. a shorter piece of DNA.
e. successful antibody secretion in response to antigen.

8. Proliferation of large pre-B cells

a. is part of clonal selection.
b. makes the pre-B cells more susceptible to apoptosis following self antigen binding
c. results in production of many B cells with the same antigen specificity.
d. results in the production of many B cells with the same VH chain but different antigen specificities due to different VL regions.
e. requires the presence of RAG-1 and RAG-2.

9. Which of the following statements is TRUE?

a. Isotypic exclusion on individual B cells pertains to expression of a single heavy chain isotype on each mature naive B cell.
b. Pre-B cells must receive a signal from specific antigen binding to pre-B receptor before they can proceed to the next stage in development.
c. Membrane m chain is always expressed with Iga and Igb
d. Membrane m chain is always expressed with VpreB and l5.
e. Transgenic mice for recombined H and L immunoglobulin genes have germline H and L genes in non-B cells.

10. Light chain rescue

a. allows self-specific B cells to repeat somatic recombination of light chain gene segments.
b. is a signal received through binding to the surrogate light chain that rescues the developing B cell from death.
c. results from multiple V-J joining events on a single chromosome until productive rearrangement of light chain occurs or all J segments have been recombined.
d. signals the developing B cell through IgaIgb to begin recombination of light chain gene segments.
e. None of the above is true.

a. does not show allelic exclusion of Ig.
b. has a mutant TdT that removes (knocks out) N nucleotides instead of inserting them during somatic recombination.
c. has no Ig gene segments.
d. has a normal gene replaced with a nonfunctional gene for the same protein.
e. is often used to produce monoclonal antibody.

16. Knock-out mice which have m gene segments from which the membrane domain has been removed

a. cannot synthesize any m chain.
b. can splice the Cm segments to the membrane segment for d and express normal amounts of membrane m chain.
c. have normal pro-B cells but no later stages of developing or mature B cells.
d. have normal pro-B cells and pre-B cells but no mature B cells.
e. have no cells with rearranged Ig genes.

17. B cells which express Iga with a truncated cytoplasmic domain (lacking the ITAMS)

a. become "stuck" in the immature B cell stage of development.
b. can express pre-B cell receptor but not proceed to divide and then recombine light chain genes.
c. develop normally since Igb has its ITAM sequences.
d. express both IgM and IgD but cannot be activated by antigen.
e. require more antigen for activation.

18. The ability of an individual B cell to express only one H chain allotype is called

a. allelic exclusion.
b. co-dominant expression.
c. isotypic exclusion.
d. nonproductive rearrangement.
e. survival of the fittest.

19. Once they leave the marrow, in order to survive mature naive B cells must enter the

a. circulation
b. lymph microenvironment.
c. primary lymphoid follicles.
d. secondary lymphoid follicles.
e. thymus.

20. Negative selection of B cells occurs when

a. immature B cells bind self antigen and undergo apoptosis.
b. immature B cells fail to bind self MHC and die.
c. lymphoid progenitor cells become committed to becoming T cells and leave the marrow for the thymus.
d. mature B cells fail to bind antigen in the lymphoid follicles and die.
e. pre-B cells bind ligand with their pre-B receptor and stop rearranging H chain genes.

21. A mature lymphocyte which has specific antigen receptors but cannot respond to that antigen is called

a. anergic.
b. apoptotic.
c. deleted.
d. lazy.
e. memory.

22. Immature B cells which bind soluble self antigen

a. become apoptotic.
b. escape clonal deletion and can potentially cause autoimmune disease in adults.
c. go on to express normal levels of IgM and IgD but cannot respond to self antigen.
d. undergo clonal deletion in the bone marrow.
e. usually cannot enter the primary lymphoid follicles and soon die in the periphery.

23. Immature B cells from inbred mouse strain q (expressing the q allele of every MHC molecule: D^q, K^q, L^q, IA^q, and IE^q) which also express transgenic H and L chains specific for H-2 IA^s (the s allele of Class II MHC IA) will undergo clonal deletion when

a. exposed to macrophages from an "s" strain mouse (expressing the "s" allele of every MHC molecule).
b. exposed to T cells from an s strain mouse.
c. exposed to their own macrophages.
d. exposed to their own T cells.
e. Both 1 and 2 are correct.

a. anergized B cells.
b. immature B cells.
c. mature naive B cells.
d. memory cells.
e. plasma cells.

26. Receptor editing

a. allows self-specific B cells to repeat somatic recombination.
b. is responsible for allelic exclusion of Ig on individual B cells.
c. kills self-specific B cells before they leave the marrow.
d. occurs during the small pro-B cell stage of development.
e. occurs only for light chains of self-specific B cells.

27. B-1 B cells are more _____________ than conventional B (B-2) cells.

a. antigen-specific
b. high affinity
c. likely to be protein-specific
d. numerous in adults
e. polyreactive

28. The antibody secreted by B-1 B cells often

a. binds common bacterial carbohydrates.
b. has a high affinity for antigen.
c. is IgG.
d. neutralizes common viral receptor proteins.
e. neutralizes self antigens.

29. As cells become mature naive B cells, they

a. become more antigen-independent.
b. leave the marrow and never return.
c. stay in one location and wait for antigen and T cells to come to them.
d. stay in the same locations as T cell to be able to receive helper signals.
e. use CAMs to enter the appropriate lymphoid organ locations.

30. A germinal center is where B cells

a. become mature.
b. divide in response to antigen.
c. first bind antigen.
d. secrete antibody.
e. undergo somatic recombination.

31. If you wanted to check for the presence of memory B cells from your vaccine subjects by taking some of their B cells and re-stimulating them in vitro (outside the body) with antigen, the best place to get some memory cells would be from

a. blood.
b. bone marrow.
c. lymph nodes.
d. spleen.
e. tonsils.

32. Multiple myeloma is a cancer which has arisen from a

a. B-1 B cell.
b. lymphoid progenitor.
c. mature B cell.
d. plasma cell.
e. pre-B cell.

33. If a B cell tumor is monoclonal, it can be differentiated from normal B cells of the same person by its unique membrane Ig

a. allotype.
b. H chain.
c. idiotype.
d. isotype.
e. L chain.

1. Cells were removed from the bone marrow of an adult mouse and incubated with FITC-anti mouse m chain and PE-anti mouse d chain. Draw the flow cytometric representation of the data. What cell types are in each quadrant of the graph: stem cells, lymphoid progenitors, myeloid progenitors, pro-B cells, pre-B cells, immature B cells, mature B cells, neutrophils, macrophages? What would be the pattern of fluorescence you would see if you stained spleen cells with the same antibodies? Are the kinds of cells in each quadrant the same? HINT: consider all the kinds of leukocytes which are found in the spleen. How would this pattern change if you used mice lacking RAG-1 and RAG-2?