Recombination and the Antibody Repertoire
Psychologists refer to the ability of a person to pick out a particular voice or word from a cacophony of sound as the "cocktail party effect." Without even trying, our minds filter through the noise, and we hear only those details that are most relevant to us.
The ability of T and B lymphocytes to recognize antigenpieces of protein or carbohydrate that stimulate an immune responseis the cellular equivalent of the cocktail party effect. Through the noise, the T and B cells in the bloodstream only recognize those antigen "words" for which they are highly specific before they spring into action. To take the analogy further, the bloodstream is a huge cocktail party with millions of circulating T and B cells, and each one only recognizes but a single word.
What enables a great number of foreign antigens to be recognized by the immune system is the extraordinarily large T and B cell "repertoire" that the body produces and maintains. This diverse repertoire is generated within cells by rearranging the appropriate genes during development and expressing these rearranged genes as receptors on the cells' surfaces. Every mature T and B cell has its own single receptor of unique specificity.
Associate Professor Ann Feeney, who is a member of the Department of Immunology at The Scripps Research Institute (TSRI), studies the factors that control the development of this repertoire and establish the diversity of T and B cells.
Diversity Through Recombination
When B cells develop from stem cells in the bone marrow, genes are rearranged that encode for large receptors the cells express on their surface to recognize an antigen and produce antibody that is specific for this antigen once it is recognized. The rearrangement brings together three segments (termed V, D, and J for variable, joining, and diversity, respectively), which are spliced together in a process that is appropriately named V(D)J recombination.
V(D)J recombination is an important source of antibody diversity, since the V(D)J segment is the part of the antibody that recognizes antigen. There are multiple copies of the V, D, and J genes in the human genome, and a functioning antibody will be one of over a million possible combinations. The final sequence is permanently spliced together so that a mature B cell will produce only one specific antibody.
The antibodies that a B cell produces will then, naturally, bind to the antigen for which they are specific, blocking viruses or bacteria from infecting cells and marking these foreign invaders for destruction by macrophages.
Since the antibody repertoire is so important for our survival in a world filled with pathogens, it is not surprising that its developmentand the process of V(D)J recombinationare tightly controlled. Accessibility of the genome during recombination is tightly regulated so that only the right genes are allowed to rearrange in the right cells (for example, only antibody genes rearrange in B cells and only T cell receptor genes rearrange in T cells).
Inappropriate V(D)J recombination is potentially dangerous because the recombinase enzyme, which is one of the crucial enzymes involved, makes breaks in DNA to initiate the process of rearranging and recombining the V, D, and J gene segments. Such DNA breaks could cause the cells to die if not properly repaired or could lead to the genes recombining in inappropriate ways.
Aberrant joining can be responsible for many "translocations," for instance, which join an immunoglobulin or T-cell receptor gene segment to a potentially lethal cancer-causing oncogene, leading to uncontrolled growth. These translocations are implicated as the cause of several leukemias and lymphomas, and Feeney hopes her studies on the control of the V(D)J recombination mechanism shed light on how the misregulation of control causes the translocations and the cancers.
And another compelling question, scientifically, is exactly how cells control V(D)J recombination and the development of the antibody repertoire.
Recombination Signal Sequences and the Navajos
One of the ways to begin to understand the mechanisms that control V(D)J recombination, suggests Feeney, is to look at the effect of those mechanismsthe recombined V, D, and J genes in the end product. She accesses these end products by looking at the recombination of genes in developing B cells in the bone marrow, asking which genes are being used, and then comparing those being used to the number of V genes in the genome.
Significantly, she says, "We've determined in the past that not all genes are used equallysome are used more than others."
Feeney and her colleagues have been focusing on short stretches of DNA that flank gene segments that are rearranged during V(D)J recombination. These "recombination signal sequences" (RSS) are the sites where the enzymes that splice the DNA bind. These RSS DNA segments are themselves variable. They are composed of conserved seven and nine base pair stretches separated by an additional 12 or 23 base pairs.
Feeney's hypothesis is that natural variations in the sequence of the RSS help explain the non-random V gene selection. Some RSS might bind the recombinase enzymes better than others, and therefore a V gene that appears more often might have a better, more effective RSS.
She tests the efficacy of various RSS segments by putting, for instance, two V genes with different RSS segments at a time into bacterial plasmids and letting them compete for rearrangement with one J gene. She then looks at which one is better by measuring the frequency of rearrangementsomething that can be easily gauged through molecular biology. She has found that sequence changes in RSS can make a gene rearrange as much as eight times less frequently in her assay, which is evidence that rearrangement may be controlled on a gene segment by the segment itself.
In support of her hypothesis, Feeney points to infections of Haemophilus influenzae, one of the leading causes of meningitis, a disease that can sometimes be fatal. Infections with this bacterium cause permanent hearing loss in one out of every five children. Like all bacterial infections, H. influenzae infections are controlled by antibodies that specifically bind to the surface of the bacteria. In antibody responses to H. influenzae , one particular antibody light chain predominates in the immune response.
Feeney and her colleagues found a polymorphisma distinct genetic variationof the RSS of this light chain gene in individuals of Navajo heritage. Navajos have a very high susceptibility to fatal H. influenzae infections, and she thinks that this "ethnic susceptibility" comes from the polymorphism in the RSS, which renders the light chain gene unable to effectively rearrange.
Location, Location, Recombination
However, Feeney and her group know that RSS is not the whole story, and they have shown that there are additional factors that control non-random gene usage besides RSS variation.
"Chromosomal location," she says, by way of example, "also can play a role [in recombination frequency]."
Her group made a discovery over a year ago that genes on the same chromosome with identical RSS can still rearrange at different frequencies, suggesting that the location of a gene within the cluster of V genes on the chromosome plays an important role in its frequency of rearrangement.
Feeney and her colleagues also published a paper last year describing another cellular mechanism that appears to influence non-random gene usage. In the study, published in the Journal of Experimental Medicine, Feeney and her colleagues showed that certain transcription factors, called E2A and EBF, which are essential for lymphocyte development, play an important role in inducing rearrangement of individual genes.
Transcription factors are DNA-binding proteins containing structures that fit into the grooves of DNA's double helix like a glove. Significantly, transcription factors regulate the expression of genes into mRNA by binding to particular sequences of DNA necessary for transcription.
They found that different V genes that were closely located on the same chromosome rearranged at different frequencies when E2A or EBF were expressed. The fact that each transcription factor induced only a subset of genes to rearrange suggests that some were made more accessible to rearrangement than others after exposure to the transcription factor. This leads Feeney and her colleagues to suggest that rearrangement is also controlled at the level of the individual genewith particular binding motifs within the genes themselves enhancing their accessibility and facilitating their own recombination preferentially.
They are currently looking for changes in the chromatin structurethe complex of protein and DNA into which genomes are packagedsurrounding genes before and after the genes become accessible for rearrangement as clues to the molecular mechanism of accessibility.
"We have learned a lot in the past few years about the mechanism of V(D)J recombination and how it makes a diverse antibody and T cell repertoire," says Feeney. "[But] we are just beginning to unravel the mysteries of how these important genes are regulated and controlled during the development of lymphocytes."