Recombination and the Antibody Repertoire
By Jason Socrates
Bardi
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 antigen—pieces
of protein or carbohydrate that stimulate an immune response—is
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 development—and the process of V(D)J recombination—are
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 mechanisms—the 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 equally—some 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 rearrangement—something
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 polymorphism—a distinct
genetic variation—of 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 gene—with
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
structure—the complex of protein and DNA into which genomes
are packaged—surrounding 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."
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