Humoral antibodies

Antibodies synthesized and released by plasma cells directly mediate humoral immunity. As shown in Figure 41-2, the basic immunoglobulin structure consists of two heavy chains and two light chains covalently linked by interchain disulfide bonds. Both chains consist of two or more domains, each defined by a single intrachain disulfide bond. The heavy chain is composed of three or four constant domains and one variable domain. The light chain incorporates one constant and one variable domain. The relatively flexible hinge regions found in certain immunoglobulins are believed to be remnants of primordial constant domains. Terminal sequences on the amino end of each chain make up the variable regions of the molecule. Within each variable region are hypervariable sequences that are responsible for specific antigen binding. There are two types of light chains, λ and κ. The ratio of λ and κ chains differs in various species, being 1 : 20 in mice and 1 : 2 in humans. Sometimes the ratio of λ to κ is used to identify multiple myeloma.

FIGURE 41-2 Diagram of an IgG antibody, including disulfide linkages. The “crystallization fragment” (Fc) of the molecule, formed by portions of the two heavy chains, contains the binding sites for specific cells and for complement; each remaining “antibody fragment” (Fab), which consists of one light chain and the remaining portion of one heavy chain, includes the variable regions (VL and VH) that participate in antibody binding. The hypervariable sequences of the variable regions are shown as thickened segments.

The class of an antibody is determined by its heavy chain. There are five heavy chains or isotypes: IgM, IgG, IgD, IgA, and IgE. IgG is the most abundant subtype and has several subclasses: IgG1, IgG2 (IgG2a and IgG2b), IgG3; and IgG4. The amino terminal region in the variable domain (v domain) of the heavy and light chains (V_H and V_L) binds to antigen, whereas constant domain (C domain) of the heavy and light chains (C_H, C_L) makes up the constant regions.

These domains form the Fc region of the molecule (Fc is the “crystallizable fragment” of a polyclonal immunoglobulin). The Fc region dictates the specific binding of each isotype to different Fc receptors on phagocytes, mast cells, and other cells involved in inflammatory reactions. The Fc region also dictates complement activation by IgG and IgM.

There are three discrete regions in the V_H and V_L domains of the antibodies named HV1 (hypervariable 1), HV2, and HV3 (HV3 is the most variable site of each domain). These are separated by structural framework regions (FRs), namely FR1, FR2, FR3, and FR4. The six hypervariable regions in each arm of the antibody that form the antigen binding site when brought together are complementary to the antigen, thus they are called complementarity determining regions or CDRs. There are three CDRs, CDR1, CDR2 and CDR3. Antigen molecules contact antibody over a broad area of its surface and binds noncovalently through electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic interactions.

Transmembrane and secreted forms of Igs are generated through alternative splicing of heavy chain domains. There are two polyadenylated sites that dictate the generation of transmembrane or secreted forms of Igs. All B cells initially express IgM in its transmembrane form, but upon contact with antigen they generate the secreted forms and undergo isotype switching to generate other antibodies.

Sequence differences between Ig heavy chains cause the various isotypes to differ in the number and location of their disulfide bonds, the number of attached oligosaccharide moieties, the number of C domains, and the length of the hinge region.

IgM, which is co-expressed with IgD on the surface of cells, predominates in neonates and during initial, or primary, immune responses to antigenic challenges. IgG, IgA, and IgE are important in responses to antigens on secondary exposure. Because of an extended half-life (approximately 21 days), IgG constitutes approximately 75% of circulating immunoglobulins. IgG is the principal opsonic antibody important for phagocytosis; it plays a central role in immune responses against submucosal antigens. Fc receptors for IgG are found on neutrophils, monocytes, and dendritic cells.

On a daily basis, approximately three times more IgA is produced than all other immunoglobulins combined. IgA has two subclasses, IgA1 and IgA2. The IgA2 isotype is found in most mucous secretions and constitutes about 40% of total salivary IgA. It is important in mucosal immunity and caries immunology. Both IgA and IgM are bound by the polymeric immunoglobulin receptor (pIgR), which is not within the Fc receptor family. Found on the basolateral surface of ductal epithelial and intestinal epithelial cells, the pIgR enables the two immunoglobulins to be transported by transcytosis across the epithelium and into secretions. IgA is important in specific immunity against supramucosal antigens and in anti-inflammatory reactions below the mucosa. Although circulatory IgA production (mostly IgA1) is equal to that of IgG, it has a shorter half-life (approximately 7 days) than IgG and a lower plasma concentration.

IgE is evolutionarily related to IgG in that both their ancestries can be traced back to IgY, the predominate inflammatory immunoglobulin found in nonmammalian vertebrates. IgE is bound with extremely high affinity by Fc receptors found on mast cells and basophils; it promotes immediate inflammation (which is important in initiating acute and chronic inflammation). IgE is also bound by low-affinity receptors present on eosinophils and enables these cells to exert anthelmintic and antiparasitic effects. IgE production is tightly controlled.

The role of IgD is less clear; it is not a secondary response antibody because it can be co-expressed with IgM on a single B cell. IgD and IgM mRNA is produced as a single transcript. Post-transcriptional modifications dictate whether IgM or IgD will be translated. IgD is believed to prevent the induction of B-cell tolerance, which can occur in B cells expressing IgM alone. Fc receptors for IgD have been found on T cells.

Each plasma cell produces a unique antibody because of the clonal distribution of the BCR. Initially, the differentiation of B cells to plasma cells results in the production of IgM antibodies. If antigen has also been presented to CD4+ T cells by B cells, the T cells can guide B-cell differentiation along the memory pathway as opposed to the plasma cell differentiation pathway. In the memory pathway, the isotype may change. Secondary antigen exposure can elicit IgA, IgG, or IgE production. This process of isotype switching promotes a more appropriate interplay of antibodies with complement and with myeloid immune cells (e.g., neutrophils, monocytes, mast cells, and eosinophils).

Initiation, Progression, and Termination of Specific Immune Responses

The immune system is normally engaged in the homeostatic regulation of host-derived antigens. Once an antigen receptor is formed it has to be rigorously tested against self peptides. Given the incredible number of receptors formed it is important that those lymphocytes which reach maturity are likely to be useful in recognizing foreign antigens. In general, developing lymphocytes whose receptors interact weakly with self-antigens, or bind antigen in a particular fashion, receive a survival signal. In contrast, lymphocytes with strongly self-reactive receptors must be deleted to prevent auto-immunity through negative selection. The fate of lymphocytes in the absence of any signal is death. In addition, the immune system participates in its more widely appreciated inflammatory function known as the immune response.

Immune responses are the measurable alterations in immune system activity after an antigenic perturbation. They are usually initiated by an immediate inflammatory reaction resulting from the activation of soluble factors (e.g., complement) found within the extracellular fluid or mediators released by resident leukocytes, especially the mast cell. The immediate inflammatory response signals postcapillary venule endothelial cells to recruit the appropriate acute-phase or chronic-phase leukocytes from the blood. Initially recruited are cells that do not need to progress through proliferation or differentiation to exert an effect, such as neutrophils. Neutrophils are the predominant cell in acute inflammation. Acute inflammation may be followed by slower, chronic inflammation involving less mature cells capable of adaptive cellular differentiation (with or without proliferation). The proliferation and differentiation of clones of cells that recognize the antigen specifically constitute the specific immune response.

Specific immune responses involve a series of events (Figure 41-3), each of which offers a potential site for immunotherapeutic intervention. Included in this series are antigen processing and presentation, T-cell selection, lymphocyte differentiation, effector function, and termination. These events occur in response to changes in the concentrations of antigens intracellularly and extracellularly.

FIGURE 41-3 Overview of specific immune responses. For simplicity, the IL-2 receptor and the TH17 and Treg helper cells are not shown. APC, Antigen-presenting cell; CMI, cell-mediated immunity; CTL, cytotoxic T lymphocyte.

Differentiation

After antigen recognition by TCR and BCR, intracellular signaling events allow the T cell to differentiate into functionally mature cells (Figure 41-4). Much present-day immunotherapy is aimed at this stage of the specific immune response. Binding of antigen to its receptor leads to the clustering of antigen receptor on lymphocytes, which is the first step in signal transduction, Clustering of antigen receptor leads to activation of intracellular signaling molecules. Protein tyrosine kinases are enzymes that affect the function of other proteins by adding a phosphate group to certain tyrosine residues. Specific growth factors such as c-kit have cytoplasmic domains that contain intrinsic tyrosine kinase activity. T- and B-cell signaling is different from c-kit signaling since the TCR and BCR do not have intrinsic kinase domains; rather they rely on the interaction with other tyrosine kinases known as receptor-associated tyrosine kinases.

FIGURE 41-4 Intracellular mediators of T-cell differentiation and its inhibition by immunosuppressant drugs. The Map kinase pathway is not shown. CsA, Cyclosporine; DAG, diacylglycerol; Rap., sirolimus.

There are specialized areas within the membrane lipid bilayer that contain high quantities of sphingolipids and cholesterol. These areas are called lipid rafts. Signaling molecules associate with lipid rafts. Disruption of the lipid rafts inhibits T- and B-cell signaling.

Signaling through T and B cells is governed by a complex array of intracellular signaling elements, which can phosphorylate and activate other elements to transduce the signal from the receptor. The receptor-associated protein kinases are localized in the inner surface of the cell membrane and cannot activate their cytosolic targets efficiently unless they themselves are activated. Their activation and subsequent phosphorylation of tyrosines on receptor-associated chains recruits other protein tyrosine kinases, which then transduce the signal. The Src family kinases involved in T and B cell receptor signaling provide an example of this kind of signaling element.

The variant chains of the lymphocyte antigen receptor are associated with invariant accessory chains that mediate the signaling function of the receptor. The BCR is associated with Igα and Igβ invariant chains. The TCR is associated with multiple invariant accessory chains (ε, γ, δ, and ζ). Accessory chains have a structure termed the immunotyrosine activation motif (ITAM), which enables them to signal when the BCR or TCR is bound to the antigen. ITAMs are phosphorylated by src family receptor–associated tyrosine kinases, which gives them the ability to bind to the members of a second family of tyrosine kinases (Syk in B cells and Zap 70 in T cells). The enzyme activity of each src family kinase is regulated by the phosphorylation status of its kinase domain and its carboxy terminal region, each having a regulatory tyrosine residue. Phosphorylation of tyrosine in the kinase domain activates the enzyme, whereas phosphorylation at the carboxy terminal tyrosine is inhibitory. Src family kinases are kept inactive by a tyrosine kinase called CSK (c-terminal src kinase), which phosphorylates the inhibitory domain. Since the function of CSK is constitutive in resting cells, src-family proteins remain quiescent until antigen recognition, which then activates a protein tyrosine phosphatase (CD45 that removes the phosphate block from the inhibitory tyrosine and permits activation of the src-family kinases.

Antigen receptor signaling is enhanced by coreceptors that bind to the same ligand. B cell co-receptors are formed by a complex of CD19, CD21, and CD81 proteins. Src family kinases phosphorylate the ITAMs on Igα and Igβ, and these subsequently recruit Syk to Igβ. Trans-phosphorylation of each kinase by the other occurs, and the activated kinases in turn activate phospholipase C-γ (PLC-γ), guanine exchange factors (GEFs), and Tec kinases.

Similar to BCRs, clustering of TCRs and the co-receptors activates CD45, which removes a phosphate from Src family kinases (e.g., Lck) and activates their function, resulting in the phosphorylation of ITAMs. After phosphorylation, ZAP-70 is able to bind and become activated by Lck, which will initiate a signaling cascade leading to the activation of PLC-γ, GEF, and Tec kinases.

PLC-γ is known to cleave membrane phosphatidylinositol bisphosphate (PIP₂), yielding diacylglycerol (DAG) and inositol triphosphate (IP₃). DAG activates protein kinase C (PKC), which in turn stimulates nerve factor-κB (NF-κB). NF-κB usually is complexed with inhibitor proteins collectively called I-κB, which prevent its translocation to the nucleus. NF-κB is an important transcription factor for various inflammatory cytokines, and supports inflammatory aspects of the specific immune response. TNF-α, one of the cytokines whose synthesis is stimulated in part by NF-κB, increases the transcription of NF-κB and its dissociation from I-κB.

IP₃ increases the intracellular Ca⁺⁺ concentration, activating calcineurin, a phosphatase that frees NF-AT (nuclear factor of activated T cells) from a phosphate block. GEFs activate Ras, an important GTPase that activates MAP kinases. The Ras-induced MAP kinases then activate Fos and Jun kinases, which are components of the transcription factor AP-1.

In unstimulated cells, NF-AT is rendered inactive and remains in the cytoplasm by phosphorylation on serine/threonine residues. Ca⁺⁺-activated calcineurin remo/>

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