Not By Chance: Controlling Affinity Maturation - Evolution News & Views

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Not By Chance: Controlling Affinity Maturation

[Editor's Note: This is part four of a six-part series from microbiologist Donald L. Ewert responding to Kathryn Applegate, of the BioLogos Foundation, in her arguments that the vertebrate adaptive immune system illustrates the claimed creative the Darwinian mechanism. Previous parts of Ewert's response can be found at the following links: Part One, Part Two, Part Three.]

Pathogen-directed activation of the immune response
The initiation of an immune response is designed so that the cellular and molecular components that are best equipped to deal with a pathogen are engaged. There are basically three response pathways. Non-protein antigens that have repeating carbohydrate units on their surface, such as are found on bacteria, can directly activate B cells. These B cell do not go through affinity maturation or class switch since multiple binding sites on the antigen make a strong bond with the B cell and the IgM class of antibody that is produced which has five receptors per molecule.

Responses to protein antigens fall into two classes, depending on whether the pathogen is intracellular or extracellular. Since intracellular antigens such as viruses are not accessible to circulating antibody, they activate cytotoxic T lymphocytes that are best equipped to kill them. This activation is directed by the Class I MHC antigen that is attached to the antigens as they are processed in cells. Extracellular proteins, which are best dealt with by circulating antibody, activate B cells to begin the process of affinity maturation and class switch that leads to the production of a monomeric IgG class of antibody. This latter pathway requires the assistance of a class of T lymphocytes called T helper cells (TH) and the interaction of Class II MHC proteins.

The collaboration with the TH cells at this stage of B cell development is critical to insuring that the antigen it has detected is indeed foreign and not one of the host organism's proteins. The B cell obtains confirmation to proceed by packaging or "presenting" a part of the antigen it recognized in a Class II MHC molecule for the T lymphocyte to "look" at. If the antigen receptor of the T lymphocyte, the TCR (which was also generated by V(D)J recombination), recognizes this antigen-MHC complex on the surface of the B cell, it in turn signals back to the B cell telling it to further differentiate into a plasma cell which produces the antibody protein, i.e., the cell-free form of the BCR. This double-key confirmation process prevents false starts, conserves energy, and reduces the risk that antibodies will be made against "self" antigens.

Subsets of TH cells are able to direct the production of specific classes of antibodies by B cells depending on the type of antigen. TH1 cells direct the production IgG antibodies that promote the ingestion and killing of microbes whereas TH2 cells direct the production of the IgE class of antibodies to pathogens such as helminthic parasites which are too large to be ingested by macrophages. The IgE coats the helminthes for destruction by eosinophils. This specialization of the adaptive immune response is not a random process and involves a matrix of intracellular and extracellular proteins that communicate information between the cells.

How do cells and pathogens find each other?
At the cellular level, as noted above, the process is initiated by the independent recognition of an antigen by two different classes of lymphocytes, a B cell and a T cell, each of which has an independent history of development that prepares them to cooperate with each other and regulate the immune response. This ensures that any potential B cell antigen (at least for T cell-dependent antigens) is confirmed by at least two antigen receptors before the immune system commits to producing high affinity class-switched antibodies. It is thus essential that these two lymphoctes, both of which have bound to the same pathogen, are able to find each other. With several million B and T cells distributed throughout the body, this is no simple task . While the chances of two cells encountering the same antigen and then coming together are very small, the activated B and T cells are programmed to release specialized proteins (chemokines) that attract one another within the confines of particular compartments found within lymphoid organs (lymph nodes and spleen). If these encounters were left to chance alone, the adaptive immune system could not mount a response in time to defend the host against a proliferating pathogen.

Controlling the destructive effects of AID
At the molecular level, SHM utilizes highly dangerous enzymes to make un-templated changes at very precise regions of the DNA. SHM is initiated by an enzyme, activation-induced cytidine deaminase (AID), which deaminates cytidine residues in single-stranded DNA that are exposed during Ig gene transcription. The resulting mutations (uridine/guanine mismatches) are then processed by mismatch repair enzymes and error-prone polymerases that normally correct errors in the DNA. In this case, however, they cause point mutations and substitutions to increase. The latter effect is the result of a pre-programmed subversion of a natural repair process, as will be discussed below.

Due to AID's potential to alter the information contained in the genome, its expression is highly regulated and targeted to specific regions in the antigen-combining site of the immunoglobulin gene called the complementarity-determining region (CDR). A recent paper by McBride et al. identified several levels of AID regulation, concluding:

Thus, AID appears to be controlled by multiple potentially overlapping mechanisms. We speculate that this type of combinatorial regulation facilitates fine control of AID level, which is required because small imbalances in its expression can result in catastrophic effects on genomic stability.

Control of AID activity is important for the following reasons:

First, it is essential to cause only subtle changes that alter the affinity the BCR for the antigen, but not the specificity of the antigen-combining site. AID induced changes are targeted to sites in the CDRs of the V region that form the antigen-combining site, when the protein is folded. These changes slightly alter the amino acid composition of the receptor, changing its affinity for the antigen. Other regions of the V gene that form the scaffolding for the V region, called framework regions (FWR), have a low mutation rate. Mutations in the FWR would damage the structure of the antibody and cause the B cell to initiate a program of cell suicide called apoptosis.

Second, the V region which contains the CDR genes is adjacent to the constant region gene segments, which are the business end of the immunoglobulin gene. The constant region of the antibody must reliably interact with other components of the immune system (Fc receptors) to execute destruction and removal of a pathogenic agent. Therefore, this region must be spared from genetic changes to preserve the ability of the antibody-activated effector mechanisms that destroy a pathogen.

Third, enzymes like AID that alter the genetic code have the potential to wreak havoc on the information contained in the genome. Therefore, their activity must be regulated to preserve the integrity of the organism. It is remarkable that mutations can occur at an exceptionally high rate to effect a positive improvement in a specific region of the antibody but be controlled so as not to damage the B cell's ability to survive and proliferate.

Enhancing mutations by subversion of DNA repair enzymes
The molecular mechanisms involved in SHM have only recently been discovered. The targeting of the hypermutation mechanism involves two steps that determine the location and frequency of preserved mutations. The initial step targets the AID to specific sites (called mutation hotspots) that support formation of mutations in CDRs while minimizing mutations in the FWRs. This is not a random process. The next step, following the introduction of AID mutations, is the activation of DNA repair enzymes (DNA polymerases) that normally repair the damaged (deaminated) nucleotides. However, due the intricate positioning of the C and G nucleotides in the V region, the enzymes' repair activity is subverted, making the changes permanent in the CDRs and leading to an increase in the number of mutations (Zheng et al).

The intricate nature of the regulatory control of this process is presented in a paper by Zheng et al. in which they identify nine special features of IgV genes that "precisely regulate SHM while retaining a functional amino acid sequence."

Evidence is presented that IgVH genes have evolved to support the initiation of SHM by AID, but to minimize the occurrence of C-to-T-induced amino acid replacements through intricate positioning of coding strand Cs.

The complexity of these evolved biases in codon use are compounded by the precise concomitant hotspot/cold-spot targeting of both AID activity and the errors typical of Polī¨ to maximize the accumulation of mutation in the CDRs and minimize mutations in FWRs.

(Zheng et al., emphasis added. Note that the gratuitous use of the adjective "evolved" was not supported in this or any other referenced research report.)