Immunoglobulins (Igs) are key to B cell function because they serve as both an antigen receptor and a major secreted product. The variable region binds antigen and is generated by random rearrangement of gene segments to give rise to numerous specificities. The constant region (Fc) defines the isotype and mediates effector functions.
Surface Ig is the major component of the B cell receptor complex, which regulates B cell selection, survival, and activation. Secreted Ig mediates antigen neutralization and opsonization with uptake by phagocytic cells, complement activation, and cellular activation or inhibition through engagement of receptors for the Fc region of Ig.
B cells are generated from hematopoietic precursors in the bone marrow and undergo several stages of maturation and selection before becoming immunocompetent, naïve B cells that reside in peripheral lymphoid organs. After antigen activation, B cells differentiate to memory cells and Ig-secreting plasma cells.
Follicular B cells respond to protein antigens in a T cell–dependent fashion and are the major source of B cell memory. B1 and marginal zone B cells are less dependent on T cell help and display limited heterogeneity of the B cell receptor.
Autoreactive B cells are generated in all individuals. Multiple checkpoints extinguish autoreactive B cells during early and later stages of B cell development. One or more of these checkpoints is breached in autoimmune-prone individuals, leading to the increased plasma cell differentiation from autoreactive B cells.
Introduction
The immune system is composed of numerous cells that are required to generate innate and adaptive immune responses. Adaptive responses are characterized by immunologic memory generated during the first exposure to an antigen, thereby permitting a rapid response to the antigen after subsequent exposure.
B cells are lymphocytes that recognize antigens through a molecule called the B cell receptor (BCR). The BCR is a surface immunoglobulin (Ig) molecule that recognizes the antigen and is associated with two additional proteins that transduce the signal. Upon encountering its antigen, a B cell begins a process of activation that leads to antibody secretion and memory formation regulated by interplay with antigen-activated T cells, dendritic cells (DCs), soluble factors, and in some cases follicular dendritic cells (FDCs). Both T and B lymphocytes can differentiate from naïve to memory cells, but only B cells have the capacity to fine-tune their antigen receptor structure to increase specificity and affinity, giving rise to more effective antibodies. Beyond immunoglobulin secretion, B cells regulate the immune response by cytokine secretion and antigen presentation to T cells in the context of class II major histocompatibility complex (MHC) molecules.
While much of the knowledge of B cell biology has been generated in mouse models, human B cell biology is described in this chapter whenever possible.
Immunoglobulins: Structure and Function
The hallmark of a B cell is the expression of the Ig molecules. There are two forms of Igs, membrane-bound Igs and secreted Igs, that are generated through alternative messenger RNA (mRNA) splicing. Cell surface Ig, also termed BCR , contributes to B cell maturation and survival and initiates an activation cascade after contact with antigens. Secreted Igs, referred to as antibodies , are produced by B cells after antigen activation to protect the host through neutralizing and eliminating the eliciting antigen.
Structurally, Igs are composed of four polypeptide chains: two identical light (L) chains with a molecular weight of approximately 25 kDa and two identical heavy (H) chains of 50 to 65 kDa. Each of the chains contains a folding motif that is highly conserved among proteins of the immune system, the Ig domain . These domains constitute the backbone of the Ig molecule and help permit pairing of the polypeptide chains ( Fig. 13.1 ). The quaternary structure of an Ig molecule assumes a Y-shaped conformation that contains two functional moieties: two identical antigen-binding regions or variable regions, which are the arms of the “Y,” and a constant region, which is the base of the “Y.”
This definition of functional moieties derives from early studies analyzing proteolytic fragments of Ig molecules. Cleavage with papain generates two identical fragments that retain antigen-binding capacity and hence are named Fab, as well as a distinct crystalizable fragment, the constant region (Fc), that mediates immune effector functions but is unable to interact with antigen.
The antigen-binding regions are formed by pairing of the variable domain of the L chain (V L ) to the variable domain of the H chain (V H ). In contrast to the remainder of the molecule, great diversity exists in the amino acid sequence of the variable domains, which allows for a broad repertoire of Ig molecules that can recognize a wide array of antigens. Within the variable region of the Ig molecule are discrete regions, known as complementary determining regions (CDRs), that make direct contact with antigen. The amino acid sequences of the CDR are highly variable and are flanked by more conserved amino acid sequences called framework regions. The H- and L-chain molecules each contain three CDRs and four framework regions (see Fig. 13.1 ). The minimal antigenic determinant recognized by the variable regions of the H and L chains is known as an epitope, which may be a continuous or discontinuous region on a protein, carbohydrate, lipid, or nucleic acid. The presence of two identical variable regions in a single Ig molecule confers the capacity to interact with repetitive antigenic determinants present in multivalent antigens (i.e., polysaccharides) or enhances avidity by binding two separate antigen molecules containing the same antigenic determinant.
The constant region directs the Ig effector functions that mediate the killing and removal of invading organisms and both the activation and homeostasis of the immune system. Strictly speaking, the constant region is formed by the constant domain of the L chain (C L ), which is paired to the first constant domain of the heavy chain (C H 1), and the remaining constant domains of the two heavy chains (C H 2, C H 3, and C H 4 in immunoglobulin M [IgM]), paired to each other. However, the functions associated with the constant region are mediated by the constant domains of the H chain.
Immunoglobulin Heavy Chain Constant Region
The specific binding interactions that occur between the Ig variable region and antigen may be sufficient to block microbial infectivity or neutralize toxins. However, the ability to eliminate pathogens is mediated by the Fc portion of the molecule. The Fc regions of antigen-antibody complexes are made accessible to serum factors that constitute the complement cascade or to cytotoxic and phagocytic cells that mediate the destruction and removal of pathogens. In mice and humans, there are five different types of H-chain constant regions, or isotypes, designated IgM (μ), IgD (δ), IgG (γ), IgA (α), and IgE (ε) ; each is encoded by a distinct constant region gene segment present in the H-chain locus of chromosome 4 in humans or chromosome 12 in mice. Each isotype is capable of specific effector functions, and each cellular receptor for Ig binds specific isotypes and initiates a distinct intra-cellular signaling cascade. The number of C H domains, the presence of a hinge region to increase flexibility between Fab regions, the serum half-life, the ability to form polymers, complement activation, and Fc receptor binding vary among isotypes. Characteristics of the different Ig H-chain isotypes are presented in Table 13.1 . , , BCRs of different isotypes may also deliver different intra-cellular signals when activated by antigen. It should be noted that the interplay between antibodies and the cells that bear the Fc receptors extends beyond pathogen clearance and shapes the immune response by mediating activation or inhibition of specific cell types and by mediating cell death.
| Characteristic | IgM | IgG | IgA | IgE | IgD |
|---|---|---|---|---|---|
| Structure | Pentamer, hexamer | Monomer | Dimer (IgA 2 ), monomer (IgA 1 ) | Monomer | Monomer |
| C H domains | 4 | 3 | 3 | 4 | 3 |
| Serum values (mg/mL) | 0.7-1.7 | 9.5-12.5 | 1.5-2.6 | 0.0003 | 0.04 |
| Serum half-life (days) | 5-10 | 7-24 | 11-14 | 1-5 | 2-8 |
| Complement activation (classic) | Yes | Yes | No | No | No |
| FcR-mediated phagocytosis | No | Yes | Yes | No | No |
| Antibody-dependent cell mediated cytotoxicity | No | Yes | No | No | No |
| Placental transfer | No | Yes | No | No | No |
| Presence in mucosal secretions | Yes | No | Yes | No | No |
| Main biologic characteristic | Primary antibody response | Secondary antibody responses | Secreted immunoglobulin | Allergy and parasite reactivity | Marker for naïve B cells |
Immunoglobulin M
IgM is the first isotype expressed in developing B cells and the first antibody secreted during a primary immune response. It is found predominantly in serum but is also present in mucosal secretions and breast milk. Because the process that increases antibody affinity for a particular antigen (affinity maturation) has not yet been initiated during the early stages of a primary immune response, IgM antibodies usually exhibit low affinity. Their low affinity is balanced by the fact that much of the secreted IgM that exists is in pentameric form, generating multiple binding sites, providing high avidity for antigen and assisting with the binding of large, multimeric antigens. IgM also exists as a monomer and a hexamer, but only the pentameric form is linked by the polypeptide, called the joining (J) chain. The J chain allows the active transport of IgM to mucosal secretions.
Many of the biologic functions of IgM are mediated by its ability to activate the classic complement pathway. The complement cascade consists of a series of enzymes that, upon activation, mediate the removal and lysis of invading organisms. Deposition of antibody molecules or complement components on the surface of the antigen assists with phagocytosis. Proteins such as antibody and complement that enhance phagocytosis are called opsonins. Once the complement cascade has been activated, monocytes, macrophages, or neutrophils engulf opsonized particles through specific receptors present on phagocytic cells such as CD21, which recognizes fragments of the C3 complement component. Activation of the complement pathway also results in the generation of the membrane attack complex, which is composed of late complement components and directly lyses C3-opsonized pathogens. Because activation of the classic complement pathway requires exposed Fc regions to be spatially close, multimeric IgM is a potent activator of the classic complement pathway once it has bound its antigen. For example, hexameric IgM is between 20 and 100 times more potent as an inducer of complement activation than is monomeric IgM. C1q is an early complement factor that binds to IgM, as well as IgG. Immune complexes containing C1q can downregulate immune responses because C1q binds LAIR-1, a negative regulator that is present on the cell surface of myeloid cells and lymphocytes. LAIR-1, when associated with C1q, can suppress inflammation by preventing DC maturation and activation induced by Toll-like receptors (TLRs).
Immunoglobulin G
IgG is the most common isotype found in serum, constituting about 70% of the circulating antibody. IgG antibodies are usually of higher affinity than IgM antibodies and predominate in a recall immune response. Four subclasses of IgG exist in humans: IgG1, IgG2, IgG3, and IgG4. IgG1 and IgG3 arise in response to viral and protein antigens. IgG2 is the main antibody present in response to polysaccharide antigens, and IgG4 participates in responses to nematodes and is observed in patients with IgG4-related systemic disease.
All IgG subclasses exist as monomers and have a high structural similarity; however, minor differences result in distinct biologic effects. IgG3 and IgG1 are potent activators of the classic complement pathway, and IgG2 can initiate the alternative complement pathway.
All IgG subclasses engage specific Fcγ receptors (FcγRs) present on DCs, macrophages, neutrophils, and natural killer (NK) cells. The FcγRs on phagocytic cells, when cross-linked, mediate the removal of immune complexes from circulation and initiate antibody-dependent, cell-mediated cytotoxicity, resulting in the release of granules that contain perforin, a pore-forming protein, and enzymes known as granzymes that induce programmed cell death (apoptosis) of target cells. , FcγR engagement also allows the internalization and subsequent presentation of antigens in the context of class II MHC molecules.
Because IgG antibodies are the only ones that cross the placental barrier, they are critical for the survival of newborns. The transport of IgG from the maternal circulation into the fetal blood supply is mediated by the neonatal FcR (FcRn). FcRn is also responsible for the long half-life of IgG in serum by blocking IgG catabolism. FcRIIb is an important inhibitory receptor expressed on myeloid cells and B cells. Its function is discussed in more detail in the later Co-Receptors section.
Immunoglobulin A
Despite its relative low concentration in serum, more IgA is produced than all other isotypes combined. Most IgA exists as secretory IgA (SIgA) in mucosal cavities and in milk and colostrum, and only a small fraction is present in serum. Two subclasses of IgA exist in humans: IgA1 and IgA2. IgA1 is mainly produced as a monomer. In contrast, polymeric IgA2 is produced along mucosal surfaces.
Polymeric IgA exists mainly as a dimer and includes a J chain (the same chain that links pentameric IgM). It is captured by the polymeric immunoglobulin receptor (pIgR) that is expressed on the basolateral surface of the epithelial cells and then transcytosed to the apical side. Release of IgA into mucosal secretions requires cleavage of the pIgR; a fragment known as the secretory component (SC) remains associated with SIgA and protects it from the action of proteases and increases its solubility in mucus, where it neutralizes toxins and inhibits the adherence of SIgA-coated micro-organisms to the mucosal surface.
People with IgA deficiency are prone to respiratory tract and diarrheal infections, as well as an increased incidence of autoimmune disorders. The presence of FcαR on the surface of neutrophils and macrophages has been suggested to play a regulatory role in the immune system; it is possible that autoimmune manifestations in IgA-deficient patients arise from the absence of immune regulation mediated by FcαR.
Immunoglobulin E
IgE is involved in protection against parasitic infections but also triggers immune responses associated with allergic reactions. Only a small amount of IgE is detectable in serum, where it exists as a monomer. Mast cells and basophils express a high-affinity IgE Fc receptor (FcεRI) that binds free IgE. Cross-linking of the FcεR by antigen-bound IgE induces degranulation and release of histamine, proteases, lipid mediators such as prostaglandin D 2 , and leukotrienes, many of which are associated with anaphylaxis.
Immunoglobulin D
The role of IgD in the humoral response has been the subject of multiple speculations. IgD is found predominantly as cell surface Ig on mature naïve B cells. Soluble IgD is scarce in serum; however, IgD-producing plasma cells are found in tonsils and tissue associated with the respiratory tract, where IgD binds to galectin-9 on basophils and mast cells to enhance protective humoral responses and inhibit IgE-induced allergic reactions. High levels of secreted IgD can be found in patients with autoinflammatory syndrome. ,
Light Chains
Two distinct L-chain polypeptides exist, designated κ and λ. L chains contain a variable and a single constant domain. Even though two L-chain isotypes exist, there is no known function associated with the L-chain constant region. The κ chain is used more often than the λ chain in human (65%) and mouse (95%) Ig molecules.
Immunoglobulin Variable Region
The recognition of a virtually unlimited number of antigens requires a mechanism to generate Ig molecules with similarly broad specificities. The molecular basis of this process has been known for several years. The Ig molecule is encoded by gene segments within distinct genetic loci residing on separate chromosomes ( Fig. 13.2 ); the H-chain locus is on human chromosome 14, the κ-chain locus is on chromosome 2, and the λ-chain locus is on chromosome 22.
The H-chain variable region is encoded by a variable (V H ), diversity (D H ), and a joining (J H ) segment. The L chain is encoded by either Vκ and Jκ or V λ and Jλ segments; it does not contain D segments. The human H-chain locus contains from 38 to 46 V H , 23 D H , and nine J H functional genes (these numbers represent a typical haplotype but vary among individuals). The κ-chain locus contains approximately 31 to 35 V κ genes and five J κ functional genes; the λ-chain locus contains 29 to 32 Vλ genes and four or five Jλ functional genes.
Generation of Immunoglobulin Diversity
In a developing B cell, different V H , D H , and J H or V L and J L gene segments are randomly combined to generate a large number of different Ig molecules (see Fig. 13.2 ). This process, known as V(D)J recombination, occurs in the fetal liver or adult bone marrow, in the absence of antigen stimulation, and must be successful to continue with B cell maturation. Here, we present the molecular process, and later the functional and developmental consequences are discussed (see the B Cell Development section).
V(D)J recombination happens sequentially, beginning with the joining of one D H segment to one J H segment; then a V H segment will be targeted to the rearranged D H J H fragment. The absence of an in-frame recombination leads to recombination of the second allele. L-chain recombination also occurs in a stepwise manner. First, the κ locus is rearranged; in the absence of a productive κ-chain rearrangement, the λ locus undergoes recombination.
The recombination machinery is composed of specific enzymes, including recombination-activating gene 1 and 2 (RAG-1 and RAG-2). The complex recognizes recombination signal sequences (RSS) that flank the V, D, and J gene segments. These highly conserved RSSs are composed of a palindromic heptamer (seven base pairs) followed by DNA spacers that are 12 or 23 base pairs in length and an adenosine/thymidine (AT)-rich nonamer. Once the complex recognizes its target, it generates double-stranded DNA (dsDNA) breaks at the RSS sites. Next, the cellular DNA repair complex recognizes and joins the cleaved segments.
The random recombination that occurs among V, D, and J gene segments can generate a diverse Ig repertoire without the need for a large number of germline H- and L-chain genes. During H-chain recombination, nucleotides may be added at V H D H and D H J H junctions by the enzyme terminal deoxynucleotidyl transferase (TdT). These non–germline-encoded sequences are known as N additions. As long as these nucleotide changes do not disrupt the reading frame or lead to the incorporation of premature stop codons, the random addition of N sequences increases the diversity of the amino acid sequence. Imprecise ligation at the coding junctions may result in the loss of nucleotides, thereby also enhancing diversity. Finally, random pairing of the H chains and L chains further diversifies the Ig repertoire.
B Cell Development
The final outcome of the maturation process is the generation of a pool of mature B cells with a diverse repertoire of Ig specificities that can recognize foreign and pathogenic antigens without compromising the integrity of the self. Therefore the process of generation of Ig diversity is coupled with the censoring of autospecificities.
B cells, as with all cells in the hematopoietic lineage, begin with the differentiation of noncommitted CD34 + hematopoietic stem cells to lymphopoietic precursors with restricted lineage potential. These cells, known as common lymphoid progenitors (CLPs), have the potential to give rise to NK, T, and B cells. Early B cell progenitors begin to express genes for DNA rearrangement, as well as B cell program transcription factors. Multiple transcription factors act in concert, but Ikaros, E2A, EBF, and Pax5 appear to be the most important in B cell development. Pax5 is considered the master transcriptional control for B cells because it is induced in early stages of B cell commitment and plays a dual role by repressing genes required for differentiation to the myelomonocytic lineage and activating B cell–specific genes such as Ig genes, CD19, and signaling molecules.
Niches for Human B Lymphopoiesis
The development of hematopoietic stem cells (CD34 + ) into mature B cells begins in the first weeks of uterine life. By the eighth gestational week, early B cell precursors can be identified in the fetal liver and omentum. From gestational week 34 and through adulthood, the bone marrow is the primary site of B lymphopoiesis. It is unequivocally established that there are differences between the B cells that originate during fetal and adult lymphopoiesis in mice, and it is becoming clear that these differences extrapolate to human lymphopoiesis as well. B cell precursors are susceptible to estrogen, and the maturation of maternal B cells is arrested at the pro–B cell stage during pregnancy; in contrast, fetal B cell precursors lack estrogen receptors and consequently are unaffected by exposure to hormones. B cells originating during prenatal life have a bias in the usage of D H and J H gene segments, and this, along with low expression of the enzyme TdT, leads to a more restricted Ig repertoire with shorter CDR3 sequences.
Whether during fetal or adult lymphopoiesis, the maturation of B cells from CLP is contingent on the presence of stromal cells that provide both contact-dependent and soluble signals. Although the nature of the interactions provided by the stromal cells to create a lymphopoiesis-permissive environment is still largely unknown, they include both survival and proliferative signals. Interactions between chemokine CXCL12 and the integrin ligand vascular cell adhesion molecule-1 (VCAM-1) on the membrane of the stromal cells and CXCR4 and very late activation antigen-4 (VLA-4) on the early B cell progenitors are required for homing of the early B cell precursors to sites of lymphopoiesis and differentiation of B cells. The molecules interleukin (IL)-7, IL-3, and the Fms-like tyrosine kinase-3 ligand (Flt-3L) promote B cell lymphopoiesis, although IL-7 appears to be dispensable for human B cell development. Matrix molecules in the microenvironment such as heparan sulfate proteoglycan are assumed to “trap” critical soluble factors.
B Cell Ontogeny
The state of Ig gene rearrangement and the expression of intra-cellular and surface proteins define the stages of B cell lymphopoiesis into early B cell progenitors, pro-B, pre-B, immature, transitional, and mature naïve B cells ( Table 13.2 ). Once a CLP begins to express transcription factors required for B cell maturation, E2A and EBF, the cell becomes an early B cell progenitor. E2A and EBF enable transcription of the proteins involved in the recombination machinery (RAG-1/RAG-2). The beginning of D to J recombination on the IgH locus marks the progress to a pro-B stage.
| Marker | HSC | Pro-B | Pre-B | Immature | Transitional 1 | Transitional 2 | Plasma |
|---|---|---|---|---|---|---|---|
| CD34 | + | + | − | − | − | − | − |
| CD19 | − | + | + | + | + | + | + |
| CD10 | − | + | + | + | + | + | − |
| CD20 | − | + | + | + | + | + | − |
| CD21 | − | − | − | − | − | + | − |
| CD22 | − | − | + | + | + | + | − |
| CD23 | − | − | − | − | − | + | − |
| CD38 | − | + | + | + | + | + | + |
| CD40 | − | + | + | + | + | + | − |
| CD45 | − | + | + | + | + | + | + |
| CD138 | − | − | − | − | − | − | + |
| RAG-1 | − | + | + | +/− | +/− | +/− | − |
| RAG-2 | − | + | + | +/− | +/− | +/− | − |
| TdT | − | + | + | − | − | − | − |
| Igα | − | + | + | + | + | + | + |
| Igβ | − | + | + | + | + | + | + |
| Heavy chain | − | − (D H −J H ) | + (V H −D H -J H ) | + | + | + | + |
| Pre-BCR | − | − | + | − | − | − | − |
| Surface IgM | − | − | − | + | + | + | − |
| Surface IgD | − | − | − | − | − | + | − |
| Light chain | − | − | + (V κ -J κ V λ -J λ ) | + | + | + | + |
Pro-B Cells
The pro-B cell stage is defined by the rearrangement of the IgH chain gene segments and synthesis of a μ-polypeptide. Pro-B cells are dependent on interactions with stromal cells. The VLA-4 integrin and CD44 both mediate adhesion to stromal cells, are highly expressed at this stage, and are believed to be important for continued development. Pro-B cells also express high levels of B cell lymphoma 2 (Bcl-2), a molecule that protects cells from apoptosis. At the onset of the pro-B cell stage, the variable gene segments of both H- and L-chain loci are in the germline configuration but accessible to the recombination machinery. A D H gene segment on one H-chain chromosome rearranges with a J H gene segment residing on the same chromosome, often with the inclusion of nontemplate nucleotides at the junction of these two segments. Next, a V H rearranges to the D H J H gene segment. Completion of V H D H J H gene rearrangement leads to the generation of an H-chain transcript that also contains the IgM constant region (C μ ), which is the constant region gene segment most proximal to the variable region gene segments on the chromosome (see Fig. 13.2 ).
The generation of a μ-polypeptide and its subsequent expression on the surface of the cell, together with a surrogate L chain formed by the λ5 and Vpre-B polypeptides, as well as the Igα/Igβ dimer—a complex known as the pre-B cell receptor (pre-BCR)—marks the end of this phase of gene recombination. This constitutes a critical developmental checkpoint and the entrance to the next developmental stage, known as the pre-B cell. The requirement for a pre-BCR complex ensures that B cells without a productive H chain will die.
The pre-BCR stimulates pro-B cells with productive IgH rearrangement to proliferate. It also transduces a signal that the V(D)J rearrangement was successful and halts recombination of the second H-chain allele. This process, known as allelic exclusion, ensures that all Ig molecules generated within a single B cell are identical and have the same antigenic specificity. If no μ chain is generated, rearrangement is initiated on the other chromosome. If the second rearrangement also results in a nonproductive H-chain molecule, the absence of a pre-BCR–mediated signal induces apoptosis. The odds of generating a productive rearrangement are one in three, and consequently, approximately 50% of the cells that begin recombination will be unable to proceed along a developmental pathway.
Pre-B Cells
The pre-B cell stage is characterized by L chain recombination. Initiation of this stage requires the presence of the pre-BCR and functional signal transduction machinery.
At the transitional stage from pro-B to pre-B cells, the expression of the pre-BCR induces a proliferative burst that generates daughter cells with the same H chain and potential for multiple specificities within daughter cells, each of which may produce a different L chain. Targeted disruption of genes encoding the pre-BCR complex such as the IgM transmembrane constant region domain, λ5, or the Igα and Igβ accessory molecules arrest B cell development. In addition, defects in the adapter molecule BLNK or the tyrosine kinase Btk lead to a serious impairment in pre-B cell maturation. It appears that the charged residues on pre-BCR induce self-aggregation, which activates constitutive internalization and signaling of the pre-BCR complex that leads to clonal expansion of B cell precursors with a fit pre-BCR.
The expression of the pre-BCR is transient. After the proliferative burst, the μ H chain is present only in the cytoplasm as the pre-B cell rearranges an L chain. The general rearrangement process is similar to V(D)J rearrangement and is dependent on RAG-1/RAG-2 expression. Because TdT is not expressed at this stage, L chains do not usually contain N sequences at the V L J L junction. At the end of this process, pairing of the newly minted L chain with the μ H chain leads to the surface expression of an IgM molecule, complexed with Igα and Igβ, to form the BCR complex. It is believed that surface expression of the BCR on immature B cells transduces signals that enforce allelic exclusion at the L-chain locus and downregulate expression of the RAG genes. This immature B cell has completed the gene rearrangement process and is now subject to repertoire selection.
Immature B Cells
Once B cells express surface IgM in the bone marrow, they are subject to repertoire censoring. During this stage, cross-linking of the BCR by antigen leads to the activation of one of several tolerance mechanisms to diminish the fraction of autoreactive cells present in the mature repertoire. These mechanisms include deletion, receptor editing, and anergy (see later discussion on negative selection).
During the maturation process in the bone marrow, the cells become less dependent on interactions with the stroma and move toward the sinusoidal lumen. Once they express IgM, they enter the blood, where they are called transitional cells.
Peripheral Naïve B Cell Subsets
As B cells mature and their dependence on stromal cells decreases, they leave the bone marrow and finish their maturation in the spleen before homing to other lymphoid tissue such as the lymph nodes, tonsils, and Peyer’s patches of the intestine. It is in these secondary lymphoid organs where mature B cells interact with foreign antigen and specific humoral immune responses are activated.
The cytokine milieu surrounding the B cell is diverse and spatially and temporarily regulated. Two members of the TNF family, BAFF and APRIL, have emerged as key survival factors, particularly at two regulatory points in development and differentiation: the transition from an immature to a naïve B cell in the periphery and the survival of the newly produced plasma cells. BAFF (B cell–activating factor, BLyS) and APRIL (a proliferation-inducing ligand) are proteins produced by cells that take part in the innate response such as macrophages and DCs, as well as stromal cells, and are present as membrane-bound proteins or soluble trimers. They have three known receptors (BAFF-R, TACI, and BCMA) that are present on the membrane of B cells from the T2 stage to their final differentiation to plasma cells. BAFF binds the three receptors, whereas APRIL binds only TACI and BCMA. BAFF induces survival and activation of B cells when bound to BAFF-R, whereas BAFF signaling through TACI decreases the size of the B cell pool. APRIL does not participate in B cell homeostasis but seems to be critical to the survival of plasmablasts in the bone marrow.
Enhanced survival and activation of autoreactive B cells have been demonstrated in mice that overexpress BAFF. An increase in serum levels of BAFF has been observed in some patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren’s syndrome and is thought to contribute to pathogenesis because autoreactive B cells that would normally be censored can survive in the presence of excess BAFF ( Fig. 13.3 ).
Transitional B Cells
Once immature B cells egress from the bone marrow, they are called transitional cells. These cells are the earliest B cells found in the periphery in healthy subjects and move to the spleen to finish their maturation.
Transitional cells are the last B cell subpopulation that expresses the developmental marker CD24. At this stage B cells begin to express surface IgD, which harbors the same specificity as the IgM because the IgD H chain is encoded by the same VDJ fragments as IgM but expresses the C δ instead of the C μ domain. It is the expression of IgD that separates transitional cells into two different maturation stages. Transitional 1 (T1) B cells that do not express IgD are the recent bone marrow emigrants, and transitional 2 (T2) B cells that begin to express IgD represent the subsequent maturational stage. The existence and functional characteristics of a third transitional stage (T3) are still debated.
Transitional cells constitute a stage subject to multiple regulatory processes. First, transitional cells must compete with naïve B cells already present in the periphery for a developmental niche. Transitional B cells are extremely dependent on BAFF. In its absence, B cell development does not progress beyond the T1 stage. Second, T1 transitional cells are still highly susceptible to tolerance induction after BCR cross-linking. In T1 cells, cross-linking of the BCR ex vivo leads to cell death, whereas T2 cells respond to BCR cross-linking by proliferation and differentiation to the mature naïve B cell stage.
Mature B Cells
The final stages of maturation that occur in the spleen and give rise to the naïve B cell subset have not been fully elucidated, but the prevailing theory is that T2 B cells give rise to the circulating mature naïve cell population. In the mouse spleen, two populations of phenotypically and functionally different naïve B cells are recognized: follicular and marginal zone (MZ) B cells. Human naïve B cells constitute 60% to 70% of the circulating B cell repertoire and populate the spleen and lymph nodes. They include the equivalent of mouse follicular B cells and represent the circulating, nonantigen-exposed B cell subpopulation characterized by surface IgM and IgD expression, lack of CD27, and the presence of the membrane transporter ABC. There is also a population in blood of IgM + , CD27 + B cells that have been likened to MZ B cells, which do not recirculate in the mouse.
Marginal Zone B Cells
MZ B cells are a population of noncirculating mature B cells located in the MZ of the rodent spleen. In rodents, MZ B cells present clear phenotypic and functional differences from the cells present in the follicles, responding to blood-borne pathogens and to repetitive antigenic structures such as the ones present on polysaccharide antigens.
In the human spleen, the structural definition of the MZ does not correspond exactly to the area surrounding B cell follicles. However, there is a population with the functional characteristics of mouse MZ B cells: low activation thresholds, highly responsive to polysaccharide antigens, and with a clear surface phenotype. These cells, which are sometimes named MZ-like or unswitched memory, are not restricted to the human spleen but are found circulating in the peripheral blood, as well as in the lymph nodes, tonsils, and Peyer’s patches. , They are defined as IgM high , IgD low , CD27 + , CD21 + , and CD1c + . ,
Given that these cells possess the “memory” marker CD27, it is suggested that they have experienced antigenic exposure; however, the presence of MZ-like B cells in subjects with X-linked agammaglobulinemia (a CD40L deficiency) suggests that even if antigen exposure has occurred, T cell help has not. Interestingly, MZ-like B cells, although already present at birth, do not seem to be fully functional at that time; infants up to age 2 years are particularly susceptible to infections by capsulated bacteria. This phenomenon might be due to the functional immaturity of the cells or to the lack of development of the antigen-trapping microstructure.
B1 Cells
In mice, B1 cells represent a minor population of B cells that reside predominantly in the pleural and peritoneal cavities. They are named B1 because it is assumed that they are the first population of B cells to develop during intrauterine life. Functionally, B1 cells have been characterized as self-renewing cells that possess a limited BCR repertoire and respond with low affinity to a broad array of antigens, mainly phospholipids and carbohydrate structures in the bacterial cell wall. Despite their low numbers, these cells secrete most of the natural antibodies of the organism (antibodies that appear without evidence of previous immunization) and are assumed to be the precursors of most of the plasma cells that home to the intestinal lamina propria. Phenotypically, these cells are defined as IgM high and IgD low . About 70% of them express the marker CD5.
In humans, the definition of B1 cells is still unresolved. The CD5 marker has been used extensively as a surrogate marker for B1 cells with mixed success, given that activated human B cells also upregulate the expression of CD5.
Sites of B Cell Homing and Activation
After the immature B cell stage, B cells home to secondary lymphoid organs, which contain the microenvironment and architecture necessary for the retention and activation of B cells. These organs include the spleen and lymph nodes, as well as lymphoid structures in mucosal tissue (e.g., Peyer’s patches, appendix, and tonsils). The secondary lymphoid tissue is adapted to trap circulating antigen and expose the B cells to it and to provide interactions with T cells and other co-stimulatory cells. Peripheral lymphoid tissue contains specialized antigen-presenting cells known as dendritic cells. In the Peyer’s patches of the intestines, foreign antigen is taken up in specialized epithelial cells known as M cells. Even though peripheral lymphoid tissues vary in structure and cellular organization, they all possess antigen-presenting cells and B cell–containing follicles surrounded by T cell–rich zones. As explained in the following section, antigen, T cells, and DCs are required for B cell activation and differentiation into Ig-secreting plasma cells or memory B cells.
Circulation and Homing
B1 cells typically home to the peritoneal and pleural cavities and, to a lesser extent, the spleen. Naïve B cells enter the peripheral circulation by passing through the endothelial lining of the sinusoids of secondary lymphoid tissue and recirculating throughout the follicles of secondary lymphoid tissues. The entry, retention, and recirculation of B cells through secondary lymphoid organs depend on both adhesion molecules and chemokine receptors. , First, expression of LFA-1 and VLA-4 is required for entry into the lymphoid tissue, and then the chemokine receptors CXCR5 and CCR7 direct localization within the tissue. The CXCR5 molecule is expressed on all mature B cells and mediates B cell migration to follicles in response to the chemokine CXCL13, which is produced by follicular stromal cells. These cells, in turn, are regulated by lymphotoxin made by B cells. In the follicle, the B cells scan for antigen, making contact with potential antigen-bearing cells such as FDCs, subcapsular macrophages, and DCs. If the B cell does not encounter a cognate antigen, it will exit the lymphoid organ through the efferent lymphatics in response to the molecule sphingosine 1 phosphate (S1P). Neutralization of S1P leads to sequestration of B cells in lymphoid organs.
Upon antigenic encounter, B cells are retained in the lymphoid organ because of the upregulation of CCR7. The ligands for CCR7 (CCL19 and CCL21) mediate the organization of the T cell zone and attract antigen-activated B cells to this border where cognate T cell–B cell interactions occur. In contrast, it is assumed that MZ-like B cells respond to antigen without the help of cognate T cells. In mice, MZ B cells are present exclusively in the spleen and do not recirculate; in humans they are found in the spleen and tonsils, as well as in blood. MZ B cells localize to the outer layers of the follicles, making them among the first cells to encounter blood-borne antigens. The adhesion molecules ICAM-1 and VCAM-1, as well as S1P and cannabinoid receptor 2, are responsible for sequestering MZ B cells within the marginal sinuses.
The interplay of chemokine expression and the induction of chemokine receptors play an important role in the germinal center (GC) response. The chemokine CXCL12 retains centroblasts in the dark zone during the process of somatic hypermutation and isotype class switching. CXCL13 regulates migration to the light zone, where survival and selection events are mediated by interactions with CXCR5-expressing T follicular helper (Tfh) cells and FDCs. Later, CXCL12 promotes the migration of plasmablasts to the bone marrow, where they undergo further development into long-lived plasma cells.
Mucosa-Associated Compartments
Within the mucosal tissue, the sites of induction of immune responses are distinct from the site where the effector cells reside. There are two main sites for the induction of an immune response. The first is the mucosa-associated lymphoid tissue (MALT) that includes Peyer’s patches, nasopharynx-associated tissue, and isolated lymphoid follicles, where exogenous antigen is displayed by specialized M cells that transport antigen to the follicle. The second site of induction includes mucosa-draining lymphoid nodes such as the mesenteric and cervical lymph nodes.
B cells reach these sites through the systemic circulation. Once they are stimulated by antigen and induced to differentiate, they home to the effector sites in the intestinal and respiratory lamina propria, where they differentiate into plasma cells and produce antibody mainly of the IgA isotype. An interesting characteristic of the plasma cells induced in the mucosal compartments is their selective homing to mucosal effector sites. Nasal activation leads to IgA-secreting cells with high levels of CCR10 and α4β1 integrin that home to the respiratory and genitourinary tracts in response to their ligands, CCL28 and VCAM-1. Migration to the intestinal lamina propria, in contrast, seems to be dependent on orally induced activation and subsequent expression on B cells of the chemokine receptor CCR9 and α4β7 integrin that bind to CCL25 and MADCAM1/VCAM-1, respectively.
B Cell Activation and Differentiation
Upon encountering antigen, a B cell is triggered by the BCR signaling cascade to rapidly change its metabolic program from a quiescent to an activated state with an increase in glycolysis and upregulation of nutrient transporters. Activated B cells, thus, amass energy and biomass needed for clonal expansion. The BCR signaling cascade ultimately initiates new gene expression that guides activated B cells to undergo either differentiation into memory B cells and plasma cells, or apoptosis. The end result of this process will depend on the characteristics of the antigen; the B cell subpopulation activated; the co-stimulatory signals provided by cytokines, growth factors, and T cells; and the microenvironment with its profile of nutrients and metabolites.
B Cell Receptor Signaling
The BCR complex is composed of surface Ig, noncovalently bound to a dimer formed by the molecules Igα and Igβ. The surface Ig on the naïve B cell includes both IgM and IgD. The role of surface Ig is to recognize foreign antigens; the Igα and Igβ molecules transduce the signal through their cytoplasmic tails that contain a particular amino acid sequence known as an immunoreceptor tyrosine-based activation motif (ITAM). This sequence contains two tyrosine residues that can be phosphorylated upon activation. After phosphorylation, the ITAM acts as a docking site for the Src homology-2 (SH2) domain to recruit tyrosine kinases and other signaling molecules.
BCRs on resting B cells are highly mobile within the plasma membrane, and they generate a ligand-independent tonic signal that is essential for B cell survival. , After cross-linking by antigen, BCRs aggregate and translocate to cholesterol- and sphingolipid-enriched membrane microdomains named lipid rafts. The signal transduction events that occur after BCR cross-linking are mediated by the subsequent recruitment and activation of intra-cellular kinases including Lyn, Fyn, Btk, and Syk. The most proximal event after BCR cross-linking is the activation of Lyn, which results in the activation of the phosphatase CD45. CD45 removes the inhibitory phosphates on the ITAMs of Igα and Igβ, and the activation of Lyn leads to the activation of Syk and Btk. There is evidence that ligation of CD19, an activating co-receptor of the BCR, leads to recruitment and activation of Vav, phosphatidylinositol 3-kinase (PI3K), Fyn, Lyn, and Lck. Subsequently, the tyrosine kinases Syk and Btk are activated by tyrosine phosphorylation. The phosphorylation of Syk triggers the activation of phospholipase C (PLC), PI3K, and Ras pathways. The activation of Syk appears to be absolutely critical for BCR-mediated signal transduction because Syk-deficient cell lines exhibit a loss of BCR-induced signaling. Btk also appears to be required for the activation of second messenger pathways. In some patients with X-linked agammaglobulinemia, a mutation in the Btk gene results in impaired BCR signaling at the pre-B cell stage. As a consequence, these patients have a greatly reduced number of mature B cells and generate poor antibody responses. In mice, however, a mutation in Btk leads to a disease known as X-linked immunodeficiency. B cell development is impaired at the transitional T2 stage, and B cells that do go on to maturity are unable to respond to certain T cell–independent antigens.
After recruitment and activation of the intra-cellular kinases, downstream pathways are initiated. Btk, Syk, and the adapter molecule BLNK are required for the activation of PLCγ. This leads to breakdown of phosphatidylinositol 4-phosphate to diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP 3 ) to trigger calcium release from intra-cellular stores and the subsequent translocation of nuclear factor of activated T cells (NFAT) to the nucleus. In addition, Btk activates Ras, which leads to nuclear translocation of the transcription factor activator protein-1 (AP-1). BCR cross-linking also activates mitogen-activated protein kinases (MAPKs) ( Fig. 13.4 ).
