By: Dr. Stephen Pelsue, Science Director
The functionality of an antibody comes down to how well it can recognize a specific structure and how tightly it binds to that structure. There are two properties that define antibody-antigen associations: affinity and avidity. Affinity is the specific binding strength between the epitope of the antigen and the paratope (or antigen binding site) of the antibody, while avidity is the measure of the total antibody-antigen complex. Avidity is dependent on the affinity of a given epitope as well as the valency of the antibody binding sites–an IgG has a antigen binding valency of 2 while and IgM has a valency of 10. The polyclonal nature of an immune response or the presence of repeated epitopes on an antigen also contribute to the avidity. You can think of the avidity as a measure of overall association of an antibody (any antibody) with an antigen and the affinity as a measure of a specific antibody interacting with a specific epitope.
While low affinity antibodies are involved in the initial immune response and the activation of B cells, typically antibodies required for neutralizing toxins or in the capture of pathogens are high affinity antibodies. In a previous blog post on antibody diversity we discussed mechanisms on how different antibodies are generated, but we did not discuss how specific antibodies, or more accurately; specific B cells, are selected. So let’s consider how antibody specificity is selected and improved during an immune response through the process of affinity maturation.
II. Germinal Center Structure and Function
As previously discussed, B cells differentiate in the bone marrow and mature in the spleen. The mature naive follicular B cells (Fo B cells) then reside in the B cell follicles in the spleen and lymph nodes. Upon exposure to an immunogen, Fo B cells with B cell receptors (BCRs), or membrane bound antibodies, that recognize (typically with low affinity) the immunogen become activated. The activated Fo B cells, associate with Follicular Dendritic Cells (FDC) and antigen specific T helper cells (Th cells) and form a Germinal Center (GC) inside the B cell follicle (See Figure 1). The GC is where the affinity maturation, proliferation, and plasma cell and memory cell differentiation takes place. The GC is divided into two regions: the dark zone (DZ) and light zone (LZ). Expansion of antigen specific B cells occurs in the DZ, while selection of the most “fit” B cell clones occurs in the LZ. The Th cells and the FDCs also reside in the light zone and provide presentation of antigen (FDCs) and survival signals (Th cells) to the highest affinity B cell clones. BCR modification also takes place in the GC. Somatic hypermutation (SHM) is initiated in the DZ to increase diversity through the introduction of mutations in the variable region genes during replication as the clones proliferate. Class switch recombination (CSR) results in isotype switching and takes place in the light zone. Some of the B cells that survive the selection process (see next section) migrate out of the GC and differentiate into either Memory B cells or plasma cells (antibody secreting cells). The B cells can cycle through the DZ and LZ to go through multiple rounds of expansion, modification, and selection to yield high affinity clones that generate antibodies with great specificity towards the immunogen.
III. Increasing Antibody Affinity Through SHM and Competition
As described above, the GC is an isolated niche inside of the B cell follicle that supports the expansion and selection of antigen specific clones. The process of selecting only the highest affinity B cell clones is referred to as affinity maturation (see Figure 2), which is initiated by the formation of the GC by the B cells, FDCs, and Th cells. Once the GC is established, the B cells undergo several rounds of proliferation, generally 5 or 6
cycles, in the DZ. During this process, activation induced deaminase (AID) is turned on which initiates the process of SHM. Therefore, as B cells are replicating, individual mutations will be introduced into the variable regions to increase the diversity of BCRs and potentially the binding affinity (both increased and decreased). Following the B cell expansion, the B cells migrate into the LZ, where they encounter FDCs that have antigen trapped, generally in immune complexes, on their cell surface. The B cells then have the opportunity to capture antigen from the FDC surface depending on the BCR affinity for the antigen. The B cells with the highest affinity BCRs are able to capture the antigen more efficiently. That by itself leads to a Darwinian survival of the fittest competition for antigen, however it is only the first step in affinity maturation. The antigen that is captured by the B cells BCR is endocytosed (taken into the cell) and processed for presentation through MHCII to Th cells. It turns out that the B cells that can most efficiently capture, process, and present the antigen will associate with the Th cells. The Th cells provide the survival signals for the perpetuation of that B cell clone. Therefore it is not just the competition for the antigen that selects the specific B cells, but also the competition for the attention of the Th cells that allow for survival to the next step in the process.
The B cells that cannot successfully compete for antigen or Th-mediated survival will die off through apoptosis, leaving the highest affinity B cell clones to move forward. At that point there are a few options: a few of the cells will escape the grasp of the GC and differentiate into memory B cells or plasma cells, CSR will likely induce isotype switching based on cytokines produced by the Th cells, most of the B cells (some following CSR) will migrate back to the DZ and start the process all over again. As a result of several rounds of the expansion, mutation, and selection cycle, high affinity B cell clones and antibodies are generated. In fact, this process may continue with sustained or subsequent antigen exposures, meaning the more you see the antigen, the higher the affinity or specificity of your antibody repertoire towards that antigen. This, of course, is within reason, as there are regulatory mechanisms that can suppress this process or even lead to inducing tolerance. Once high affinity B cells are generated, the plasma cells that are generated will produce antibody, and memory cells will provide rapid future responses.
The process of affinity maturation is critical for developing the high affinity antibodies necessary for an efficient and effective immune response. Understanding the principles of the affinity maturation is also important in hybridoma development. In order to generate monoclonal antibodies appropriate for research and/ or clinical applications, the specificity of the antibody must be considered. The type of antigen, dose, immunization route and protocol all have impacts on developing the immune response, including affinity maturation. Giving these aspects appropriate consideration during the development of a monoclonal antibody project will certainly improve the likelihood a successful outcome is achieved.
ABOUT DR. PELSUE
Dr. Pelsue joined MBS as Science Director after 20 years experience in Immunology research. Dr. Pelsue is providing technical expertise and leadership to address challenging new antibody targets on behalf of our client base.