Humoral Immunity

Aims and Objectives:

1. To understand the basic structure of antibodies.
2. To recognise the functions of antibodies and how different regions within the antibody molecule subserve these roles.
3. To realise the essential differences of the primary and the secondary humoral immune response, and the requirement for immunological memory.
4. To appreciate the way in which the immense diversity of antibody specificities is determined.

Humoral Immunity

Antibodies - What Are They?
Basic Structure

There are 5 classes ('isotypes') of Ig; IgM, IgG, IgA, IgD and IgE, plus 4 subtypes of IgG (IgG1-4), and 2 of IgA (IgA1, IgA2). Light chains exist in two classes, lambda and kappa. Each antibody molecule has either lambda or kappa light chains, not both. Igs are found in serum and in secretions from mucosal surfaces. They are produced and secreted by plasma cells which are found mainly within lymph nodes, and which do not circulate. Plasma cells are derived from B lymphocytes:

As seen in the diagram, the immunoglobulin molecule consists of two light chains, each of approximate molecular weight 25,000, and two heavy chains, each of approximate molecular weight 50,000. IgA exists in monomeric and dimeric form, IgM in pentameric form, MW 900,000 kd. The links between monomers are made by a J chain. Additionally, IgA molecules receive a secretory component from the epithelial cells into which they pass. This is used to transport them through the cell and remains attached to the IgA molecule within secretions at the mucosal surface. The heavy and light chains consist of amino acid sequences. In the regions concerned with antigen binding, these regions are extremely variable, whereas in other regions of the molecule, they are relatively constant. Thus each heavy and each light chain possesses a variable and a constant region. The isotype of an Ig is determined by the constant region.

L chains are separated from H chains by disulphide (S-S) links. Intrachain S-S links divide H and L chains into domains which are separately folded. Thus, an IgG molecule contains 3 H chain domains, written CH1, CH2 and CH3. Between CH1 and CH2, there are many cysteine and proline residues. This is known as the hinge region and confers flexibility to the Fab arms of the Ig molecule. This is used when antibody interacts with antigen.

Antibodies - Where Are They Made?

B lymphocytes evolve into plasma cells under the influence of T cell released cytokines. Plasma cells secrete antibodies in greater amounts, but do not divide. They exist in lymphoid tissues, not blood. Other B cells circulate as memory cells.

The Life Of The B Cell

• To interact with antigenic epitopes, using their immunoglobulin receptors.
• To subsequently develop into plasma cells, secreting large amounts of specific antibody, or
• To circulate as memory cells.
• To present antigenic peptides to T cells, consequent upon interiorization and processing of the original antigen. (This will be explained later in the course).

Antibodies - What Are Their Functions?

Agglutination of particulate matter, including bacteria and viruses. IgM is particularly suitable for this, as it is able to change its shape from a star form to a form resembling a crab.

Opsonization i.e. coating of bacteria for which the antibody's Fab region has specificity (especially IgG). This facilitates subsequent phagocytosis by cells possessing an Fc receptor, e.g. neutrophil polymorphonuclear leucocytes ("polymorphs").

Thus it can be seen that in opsonization and phagocytosis both the Fab and the Fc portions of the immunoglobulin molecule are involved.

Neutralization of toxins released by bacteria e.g. tetanus toxin is neutralized when specific IgG antibody binds, thus preventing the toxin binding to motor end plates and causing persistent stimulation, manifest as sustained muscular contraction which is the hallmark of tetanic spasms. This applies particularly to IgG. In the case of viruses, antibodies can hinder their ability to attach to receptors on host cells. Here, only Fab is involved.

Immobilization of bacteria. Antibodies against bacterial ciliae or flagellae will hinder their movement and ability to escape the attentions of phagocytic cells. Again, only Fab is involved.

Complement activation (classical pathway) especially by the Fc region of IgM and IgG, leads eventually to death of bacteria by the terminal complement components which punch holes in the cell wall, leading to an osmotic death. Complement components also facilitate phagocytosis by cells possessing a receptor for C3b, e.g. polymorphs.

Mucosal protection. This is provided mainly by IgA, and to a lesser degree, IgG. IgA acts chiefly by inhibiting pathogens from gaining attachment to mucosal surfaces. This is a Fab function.

Expulsion as a consequence of Mast cell degranulation. As a consequence of antigen e.g. parasitic worms, binding to specific IgE attached to mast cells by their receptor for IgE Fc, there is release of mediators from the mast cell. This leads to contraction of smooth muscle, which can result in diarrhoea, and expulsion of parasites. Here we see involvement of both Fab v. Parasite antigen, and Fc anchoring the reacting participants. See figure 4.

Precipitation of soluble antigens by immune complex formation. These consist of antigen linked to antibody. Depending on ratio of antigen to antibody, these can be of varying size. When fixed at one site, they can be removed by phagocytic cells. They may also circulate prior to localization and removal, and can fix complement. Here Fab and Fc are involved.

Antibody dependent cell mediated cytotoxicity (ADCC). Antibodies bind to organisms via their Fab region. Large granular lymphocytes (Natural Killer cells - abbreviated NK cells), attach via Fc receptors, and kill these organisms not by phagocytosis but by release of toxic substances called perforins.

Conferring immunity to the foetus by the transplantal passage of IgG. IgG is the only class (isotope) of immunoglobulin that can cross the placenta and enter the foetal circulation, where it confers immune protection. This is of great importance to the foetus in the first 3 months. The precise function of IgD is not known. It may serve as a maturation marker of B lymphocytes.

Primary And Secondary Response

Primary Response:

• Slow in Onset
• Low in Magnitude
• Short Lived
• IgM

Secondary Response:

• Rapid in Onset
• High in Magnitude
• Long Lived
• IgG (Or IgA, or IgE)

Thus we can see that the secondary response requires the phenomen known as class switching. This requires co-operation with T cells of various types, which release cocktails of substances called cytokines. These cytokines induce gene rearrangements culminating in class switching. Details of this will be given at other points in the course.

This phenomenon is possible because the immune system possesses specific memory for antigens. It occurs because during the primary response, some B lymphocytes, in addition to those differentiating into antibody secreting plasma cells, become memory cells which are long lived.

Antibodies - How Can They Recognize So Many Different Epitopes?
Generation Of Diversity

As mentioned before, variable (V), and constant regions (C) are genetically encoded. If we bear in mind that we need to be capable of responding to something of the order of 1018 antigens, we can appreciate the need for an enormous number of genes necessary to provide this. In fact, the amount of DNA that this would involve would be quite profligate, and nature has solved this problem very ingeniously by a neat little trick.

In the germline DNA, the V genes encoding the antigen combining sites need to combine with the C genes. Diversity of specificity is brought about by additional interposed genes. In light chains, these are the J genes, which link V to C, i.e. we have V-J-C. Joining is imprecise, causing further variation, or "combinatorial diversity". In the case of H chains, there is yet another region interposed between V and J, the D (for diversity), gene segment. Thus, in H chains, we have V-D-J-C, again with combinatorial diversity. So, if we realise that there are 25 lambda light chain V genes, and 5 J genes, constituting light chain variable regions, we see there are already 125 possible combinations, disregarding imprecision of joining. For kappa light chains, there are 5 V genes and 70 J genes, yielding 350 combinations. For H chains, there are 100 V genes, 50 D genes, and 6 J genes, giving 30,000 combinations. Overall, disregarding combinatorial diversity, this yields more than 109 combinations. When we multiply this by joining imprecision, plus a heightened mutation rate of genes in the hypervariable region, we can see that from 261 genes, we can easily exceed 1018 variations.

The constant (C) regions are also genetically encoded, there being 4 genes for lambda light chains, 1 for kappa and 9 H chain C genes (IgM, IgD, IgG1-4, IgA1, IgA2, IgE).

IgG is the only class of immunoglobulin capable of crossing the placenta (an Fc mediated event), see figure 4.

Mechanisms For Generating Antibody Diversity:

• Multiple germ line V genes.
• V-J and V-D-J recombinations.
• Combinatorial diversity (= Recombinational Inaccuracies).
• Somatic point mutation.
• Pairing of heavy and light chains.

© AJC 2007.