B Cells

B-Cell Development

Definition

B (bursa-derived) lymphocytes, or B cells, are a type of lymphocyte that arises from the common lymphoid progenitor.

• Involved in humoral adaptive immunity
• Functions: 
  • B cells differentiate into plasma cells → produce antibodies (which prevent infection by inhibiting microbes from attaching to target cells) 
  • B cells differentiate into memory cells → activated against reinfection

Development

• Begins transiently in the fetal liver prenatally and continues in the bone marrow throughout life
• In the bone marrow: hematopoietic stem cells (HSCs) → common lymphoid progenitor (CLP)
• In order to produce a functional mature B cell from the CLP:
  • Cell-surface Ig molecule (a part of the B-cell receptor (BCR)) has to be expressed.
  • Germ-line DNA does not have the complete genes encoding a complete Ig.
  • Gene rearrangements (uniting different gene segments) within B cells are needed to assemble the Ig molecule.
  • This process also produces a repertoire of diverse B cells; these, in effect, create protection against different kinds of infections.
• Cell-surface (Ig) molecule:
  • Has heavy chains (μ, δ, γ, α, or ε) disulfide-linked to light chains (κ or λ)
  • Heavy-chain genes (found within a single gene locus, Ig heavy chain (IGH)), are assembled from 4 gene segments:
    • Variable region (V)
    • Diversity segment (D)
    • Joining region (J)
    • Constant region (C)
  • The light-chain genes (found as 2 separate gene loci: the κ locus [IGK] and the λ locus [IGL]) come from 3 gene segments:
    • Variable region (V)
    • Joining region (J) 
    • Constant region (C)

Stages

To reach functionality, the B cell goes through stages in the bone marrow and the secondary lymphoid organs:

• In the initial stages occurring in the bone marrow, the aim is to build the receptor (requiring no antigen). 
• When released to the secondary lymphoid organs, an antigen (with or without T-cell help) will activate the B cell to continue the maturation process.

B-Cell Activation

The B cell migrates from the bone marrow to the secondary lymphoid organs. This process takes a number of measures to produce a functional differentiated B cell: activation by an antigen, proliferation, affinity maturation, class switching, and differentiation (into plasma or memory cell).

Initial process of activation

• Naive B cells migrate to secondary lymphoid organs, primarily the lymph nodes and the spleen.
  • In the lymph nodes:
    • B cells are in the cortex.
    • T cells are in the paracortex.
    • B-cell entry into the tissue is by binding to a specialized endothelium (high endothelial venules (HEVs)).
  • Once in the secondary lymphoid organs, surface IgM and IgD are expressed.
• The B cells are resting cells that undergo apoptosis if not activated (by antigen).
• Two signals are needed for activation of B cells:
  • Signal 1: binding of antigen to the BCR (the more BCRs cross-linked by the antigen, the stronger the signal)
  • Signal 2: 
    • Inflammatory sources or antigens present a threat to the host.
    • Without signal 2, B cells do not become activated (this prevents inadvertent activation by harmless antigens).

Clonal selection

• Antigen challenge: 
  • Interaction occurs only between the antigen and the B cell with the appropriate “fit” or best “match” (based on the specific BCR).
  • This is a form of positive selection, with the newly bound B cell activated to respond.
  • Once binding occurs, that B cell divides, forming a clone.
• The selected clone will undergo clonal expansion (or proliferation) with the help of T cells.

Types of activation

B-cell activation by antigen presentation can have different paths:

• T-cell-dependent:
  • Circulating antigen interacts with the BCR.
  • The antigen is endocytosed and degraded.
  • Then the peptide components are complexed with cell-surface MHC II molecules.
  • Role of T cells:
    • T follicular helper (Tfh) cells are specialized CD4+ T helper cells previously activated by dendritic cells (by presenting the same antigen).
    • The Tfh cells recognize and bind the antigen–MHC II complex.
    • The Tfh cells then express CD40 ligand (CD40L), which binds B-cell CD40, leading to B-cell activation and proliferation.
  • Activated B cells enter and proliferate in the germinal centers, where they continue the process, leading to differentiation.
  • An example of this is the 13-valent pneumococcal conjugate vaccine (PCV13):
    • The polysaccharide–protein conjugate induces a T-cell–dependent immune response.
    • Forms both pneumococcal serotype (PS)–specific antibodies and memory B cells, creating immunologic memory.
• T-cell-independent:
  • B-cell activation does not always need the help of T cells.
  • Some antigens, such as the polysaccharides of a bacterial cell, can directly stimulate B cells and bind many IgM receptors to achieve a strong signal 1.
  • Signal 2 can be complement C3b derivatives attached to the bacterial cell or pathogen-associated molecular patterns.
  • Bypassing T-cell help, these responses are short-lived, generated mostly by IgM production (limited class switching and no memory cells).
  • An example is the pneumococcal polysaccharide vaccine 23 (PPSV23):
    • Carries surface polysaccharides of 23 serotypes of Streptococcus pneumoniae
    • Signal 1 is the polysaccharide, and signal 2 is the adjuvant (no peptides/protein to be recognized by Th cells).
    • With the 2 signals, B-cell activation and proliferation take place independently of T cells.

B-Cell Maturation and Differentiation

Affinity maturation

• While the B cell has been activated, processes in the dark zone of the germinal center take place to further “fine-tune” the antibody affinity to the antigen.
•  Affinity maturation is the mechanism by which B cells, after repeated stimulation, increase their affinity to a specific antigen presented to them.
• Increased affinity is facilitated by somatic hypermutation (SHM):
  • A programmed mutation involving the variable regions of Ig heavy- and light-chain genes, occurring after antigen-dependent activation
  • Driven by DNA-modifying enzymes: 
    • Activation-induced cytidine deaminase (AID) 
    • Uracil nucleoside glycosylase (UNG) 
  • Produces BCR with enhanced ability to recognize and bind antigen
• Selection:
  • After mutation, B cells with high-affinity BCRs now move to the light zone and access the antigen presented by follicular dendritic cells (FDCs). 
    • High affinity with the antigen → the more likely they are selected to present to and receive survival signals from Tfh cells
    • B cells with less affinity will not receive survival signals and will die by apoptosis.
• This process not only allows diversity, but also permits only the most optimized B cells to survive and differentiate.

Class-switch recombination (CSR)

• The surviving B cells (with high affinity to antigen) next undergo class switching, a step that also requires AID.
  • The constant region of the heavy chain can change the μ segment to one of the other heavy-chain segments (γ, ε, or α). 
  • The heavy-chain makeup determines the Ig class: 
    • μ: IgM
    • δ: IgD
    • γ: IgG
    • α: IgA
    • ε: IgE
  • Switching is influenced by cytokines.
    • TGF-β: preferentially switches to IgA
    • IL-4: IgE
    • IFN-γ, IL-4: IgG
  • The constant region of Ig heavy chain is changed, but the variable region remains unchanged.
  • Since the variable region is intact, specificity of the antibody does not change.
• After class switching, B cells exit the germinal centers and terminally differentiate into plasma cells or memory cells.

Plasma cells and memory cells

• Plasma cells: 
  • Large cells (up to 20 microns in diameter)
  • Produce antibodies
  • Migrate to the bone marrow
• Memory cells:
  • React to antigenic stimulation (in response to reinfection)
  • Generate plasma cells, which have high-affinity antibodies in secondary immune responses

Antibody Diversity

From the initial B-cell production, many processes allow humans to produce different antibody molecules that are significantly more than the number of genes in the genome.

It is estimated that billions of antibodies are generated, compared to about 30,000 genes.

The immune system has unique mechanisms to create antibody diversity, which include:

• Having multiple V, D, J segments: 
  • As mentioned in the discussion of early B-cell development, the heavy chains and light chains have multiple segments.
  • V, D, J, C for heavy chain
  • V, J, C for light chain
• Rearrangements of the V, D, J segments:
  • DNA sequences (called recombination signal sequences (RSSs)) flank each gene segment.
  • These sequences are recognition sites for the joining process.
  • Recombinase enzyme complex RAG1 and RAG2 (recombination activating genes 1 and 2) recognizes the RSS and catalyzes the joining process.
  • Deficiency in RAG1 or RAG2 can produce nonfunctional B cells. 
  • ***As previously mentioned in the earlier section, after the heavy chain segments, the light chain segments are also recombined.
• Junctional diversity:
  • Joining of antibody gene segments can be imprecise.
  • A number of nucleotides can be removed and/or can be inserted from the ends of the recombining gene segments.
• Combinatorial diversity: Diversity is created by the random pairing of the heavy and light chains.
• Somatic hypermutation: 
  • Point mutations occur with repeated antigen stimulation (from primary to secondary responses).
  • Increases affinity to antigen
  • Creates additional diversity to the antibody

Clinical Relevance

• X-linked agammaglobulinemia: results from mutations in the X-chromosome gene encoding for Bruton tyrosine kinase (BTK), which is essential for B-cell development and maturation. The disease is characterized by the absence of B cells, leading to recurrent infections, primarily by encapsulated bacteria and viruses, involving the lungs, sinuses, and skin, as well as the CNS. Treatment involves the administration of immune globulin.
• Common variable immunodeficiency (CVID): also known as humoral immunodeficiency. Common variable immunodeficiency is a disorder of the immune system characterized by reduced serum levels of IgG, IgA, and IgM.  The underlying causes of CVID are largely unknown. Patients with this condition are prone to infections in the GI tract and the upper and lower respiratory tracts. Common variable immunodeficiency is also associated with a higher risk of developing autoimmune disorders, granulomatous diseases, and malignancy. The treatment is immune globulin replacement therapy.
• Hyper IgM syndrome: characterized by normal or elevated levels of IgM with decreased or absent levels of other Igs. There are X-linked and autosomal recessive types of hyper IgM syndrome. The patients present with recurrent sinopulmonary infections, chronic diarrhea, and lymphoid hyperplasia. The diagnosis is verified by genetic testing. Treatment includes immune globulin replacement therapy and prophylactic antibiotics. Hematopoietic stem cell transplantation is another option. 
• IgA deficiency: characterized by low levels of IgA, with normal IgG and IgM levels. IgA deficiency is the most common primary immunodeficiency. Many patients are asymptomatic; however, there is the potential for recurrent infections as well as autoimmune disease. Patients may be prone to anaphylactic transfusion reactions because of the presence of IgA in blood products. Some of these cases eventually progress to CVID.  Treatment involves prophylactic antibiotics and avoidance of blood products that contain IgA.