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Immunogenetics
Introduction
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How can the body identify and eliminate foreign invaders. especially
since microorganisms are constantly evolving ways to avoid
detection?
The trick is to be able to distinguish between “self” and “non-self”: to
recognize molecules in the body that don’t belong there.
Foreign molecules (often on the surface of foreign organisms) raise an
immune response in the body.
The primary defense is a set of antibody molecules (also called
immunoglobulins, Ig). The human body produces over 1,000,000
different antibodies for this purpose.
Antibody molecules bind to antigens, which are molecules that are
non-self. Each antibody is specific for a particular antigen.
Some antibodies are present in the blood and other body spaces as
soluble molecules. The antibodies cause antigens to clump up and
form precipitates that scavenger cells pick up and digest.
Some soluble antibodies mark foreign cells for attack by the
complement system, a series of proteins that punches holes in the cell
membranes.
Other antibodies are on the surface of immune system cells
(lymphocytes), which are stimulated to engulf and digest foreign cells
by phagocytosis.
Complement
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The complement system is a way of
rupturing the membrane of invading
cells.
Antigens on the surface of the invader
bind to soluble antibodies.
Then a series of other proteins
(already circulating in the blood) is
activated: the complement cascade.
These proteins bind to the complex in
a specific sequence, creating a large
hole, which usually kills the cell.
This is a compliment.
The Immune System
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The lymphocytes, or white blood cells,
mostly travel through the body in the
lymph vessels, a separate circulatory
system that is connected to the blood
system. The cells collect in lymph nodes,
where large numbers of lymphocytes can
attack foreign invaders.
There are two main branches of immune
system: T cells and B cells. Both originate
in the bone marrow, from the same stem
cells as the red blood cells.
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T cells then move to the thymus gland to
mature.
B cells were originally named for their site
of maturation in birds: the Bursa of
Fabricius. This organ doesn’t exist in
humans. B cells probably mature in the
bone marrow.
B cells secrete soluble antibodies:
humoral immunity.
T cells interact directly with their targets:
cellular immunity
In the early 1900’s, there are a major
debate as to whether the immune
response was humoral or cellular. Turned
out they were both right.
Antibody Molecules
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Basic structure: 2 heavy chains
plus 2 light chains, joined together
by disulfide bridges between
cysteine amino acids.
The molecule has a "Y" shape,
with the two ends of the fork being
composed of both heavy and light
chain regions.
These ends are the regions that
bind the antigens (Ag). Each Ab
molecule has two identical Ag
binding regions, and thus the Ab
molecules can bind together large
groups of Ag's. This makes an
insoluble complex that is easy for
other cells in the immune system
to find and eat.
More Antibody Molecules
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Each light (L) chain has 2 domains, a
variable (V) region and a constant (C)
region. There are only a small number of C
regions in each person, but there are very
many different V regions. Note that the V
and C regions are together on the same
polypeptide chain!
Each heavy (H) chain has 4 domains, a V
domain followed by 3 C domains. The C
domains determine the class (IgG, IgM, etc)
of the antibody.
Ig's come in 5 classes: IgM (early response),
IgG (main blood Ig), IgA (in body
secretions), IgE (allergic response), and IgD
(mostly a cell surface molecule in the early
response).
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IgM comes in both membrane-bound and
soluble forms. The soluble form is 5 Ig’s
connected together in a star shape.
IgA is two Ig’s connected together tail to tail.
In many cases, the constant class-specific
regions of the H chains bind to receptors on
the surface of specific cells. For instance,
IgA binds to secretory cells so it gets
secreted into tears, mucus, etc. Also, IgE
binds to mast cells that trigger histamine
release and other rapid responses to
invasion.
Generation of Antibody Diversity
• There isn't room in the genome for 1,000,000
different antibodies.
• Coding is done in pieces, with a unique DNA
splicing mechanism used to assemble the L and
H chains. Each immune system cell splices the
DNA differently.
• Thus, the DNA in the region of the H and L chain
genes in B and T cells is not identical to the DNA
in other body cells.
Light Chain Splicing
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There are two different L chain genes, called
kappa and lambda. Immunoglobulins use one
or the other.
At the kappa L chain gene, there is a single
region of DNA that codes for the C domain.
Upstream from the C domain is a group of
about 250 V domains and another group of 5 J
(for "joining") regions.
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Each V region has a 5’UTR segment, a separate exon,
attached to it: the leader.
During the development of the B cell, a
randomly chosen V domain joins with a
randomly chosen J domain to form a VJ
domain. This occurs by splicing out the DNA
between them.
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Note that this is a very different mechanism from intron
spicing that occurs in the RNA of most genes!
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Once the VJ domain is formed, it can be
transcribed into RNA along with the C domain
and any DNA that lies between the VJ and C.
All the intervening RNA is spliced out as an
intron, so the final messenger RNA has VJC
all together; this is then translated into a L
chain protein.
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Lambda light chain genes are slightly different:
fewer V regions, and four different C regions
each of which has its own J. Lambda chains
use only a single DNA splice, to join a
randomly selected V region to one of the 4 J-C
regions.
Heavy Chains
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The process of producing a heavy
chain is very similar to L chains,
except that there is an additional group
of 5 D regions between the V regions
and the J regions.
Two DNA splices occur for H chains:
first a random J joins with a random D
to form a DJ region, then a random V
region is added to form a VDJ domain.
Just as in the L chain, this VDJ is
transcribed along with the C region,
and any intervening RNA is spliced out
as an intron. The final product has
VDJC all joined together and gets
translated into an H chain protein.
All of the C regions are at the heavy
chain locus. Initially, the CM region is
transcribed. CD is also transcribed and
expressed using an alternative RNA
splicing mechanism.
Summary of Splices
• Light chain
– V-JC joining by DNA splice (lambda)
– V-J joining by DNA splice (kappa)
– VJ-C intron removal (RNA splice)
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Heavy chain
D-J joining by DNA splice
V-DJ joining by DNA splice
VDJ-C intron removal by RNA splice
IgM membrane bound 3rd C domain to soluble
3rd C domain by RNA splice
• Class switching by DNA splice
Additional Diversity Mechanisms
• In addition to the DNA splicing,
other variants in the antibody
molecules are generated by
two mechanisms:
• First, the DNA splices do not
occur at a precise point: they
can vary by several bases,
which can lead to the addition
or deletion of 1 or 2 amino
acids at each splice point.
– These variations can lead to
different specificities of the
antibodies.
– The enzymes that do the DNA
splicing (RAG1 and RAG2)
produce double stranded
breaks in the DNA, which is
repaired imprecisely.
Somatic Hypermutation
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Second, there is a “somatic
hypermutation” mechanism by which
random base change mutations
occur in the V regions in B cells.
– This mechanism doesn't work in
other cells and doesn't affect other
genes: only a region of about 1.5 kb
is affected.
– It only occurs as the B cell is
maturing: after it has been
stimulated to divide by an antigen,
somatic hypermutation occurs to
modify the antigen binding region.
– Those cells that bind the antigen
most tightly survive and divide more
than the others. This process is
called “affinity maturation”.
– It is triggered by the enzyme
“activation-induced cytidine
deaminase” (AID), which deaminates
cytidine to uracil. This base
mismatch can be incorrectly repaired
by several different mechanisms to
generate mutations.
Allelic Exclusion
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These diversity mechanisms
often generate non-functional Ig
genes: genes that contain stop
codons or don't stay in the
proper reading frame. The
developing B cells use a
mechanism called "allelic
exclusion", in which each B
cell makes only 1 active L chain
and 1 active H chain. The cell
tries each copy of the L genes
and each copy of the H genes
in turn:
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If an active chain is made, no
further DNA splicing occurs.
However, if a non-functional Ig
is made, the cell then tries the
next L or H gene.
This process continues until an
active product is made from
both H and L, or until all genes
have been tried (in which case
the cell dies).
Class Switching
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Heavy chains fall into 5 classes,
based on their C regions.
Each H gene has C regions for all
5 classes arranged on the
chromosome, with the IgM C
region nearest to the V regions.
There are several different C
regions for some of the classes.
IgM is the initial Ab made by each
B cell.
However, after a while the B cell
switches to a different class.
This is done using a third DNA
splice, in which the DNA between
the VDJ and the constant region
for the new class is spliced out.
Alternative Splicing in Early
Activation
• Before it is activated, the B cell carries IgM on its
surface. This happens because the third Constant region
domain in the IgM molecule contains a stretch of
hydrophobic amino acids that anchor it to the membrane.
• However, after the B cell is activated, it starts to secrete
IgM molecules with the same V region on their H chain
but with a different third C domain.
• This happens because there are 2 third C domains for
IgM; which one is used depends on alternate RNA
splicing: before B cell activation, the membrane-bound
domain is used, and after activation this domain is
spliced out as an intron and the soluble domain is used
instead.
T Cell Receptors
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The TCR protein has 2 subunits
and one antigen binding site.
The alpha subunit has V and J
segments (similar to Ig light
chains)
The beta subunit has V, D and J
regions, like the Ig heavy chain.
Both segments undergo DNA
splicing rearrangements like the Ig
genes. The joining is not precise
and short additions or deletions of
bases can occur, as in the Ig
genes. However, affinity
maturation and somatic
hypermutation do not occur.
The TCR protein is membrane
bound. It is only found on T cells.
MHC Proteins
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Class 1 MHC molecules are found on the surface of all
nucleated cells. They are involved in cellular immunity.
Class 2 MHC molecules are only found on the surface of
cells that display antigens: macrophages and B cells. They
are involved in humoral immunity.
Structure: Both class 1 and class 2 molecules have 4
domains, but they are divided differently. MHC1 has 3
domains on the alpha subunit and one domain on the beta
subunit. MHC2 has two domains on both alpha and beta
subunits.
The peptide-binding groove is between the alpha-1 and
alpha-2 domains on MHC1, and between alpha-1 and beta1 in MHC2. Most of the differences between the many
different MHC alleles lie in this region, which allows binding
of different peptides.
The MHC proteins are encoded by a series of genes at the
MHC locus on chromosome 6. MHC genes are well known
to be very polymorphic. This polymorphism is the major
cause of tissue graft rejection: the immune system almost
never recognizes MHC genes from another individual as
self.
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There are three main MHC class 1 genes: A, B, and C, which
encode the alpha subunit. The beta subunit (beta2
microglobulin), is not very polymorphic, and it is encoded
elsewhere. Also, there are about 25 “non-classical” class 1b
genes and pseudogenes clustered nearby. The class 1b genes
are mostly monomorphic and their function is not well
understood.
The class 2 genes are encoded by 6 regions: DM, DN, DO, DP,
DQ, and DR. Each region has at least one alpha and one beta
gene. There are also some other genes and pseudogenes in
the region.
Activation of B Cells
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In the embryo, each B cell undergoes the
DNA splicing necessary to produce a single
type of antibody. This antibody is an IgM
bound to the surface of the cell. No
antibody is secreted at this time.
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Foreign invaders are swallowed
(phagocytosis) by macrophages, which are
lymphocytes that non-selectively eat
particles found in the body.
The macrophage partially digests the
invader, converting it into peptides and other
small molecules.
The macrophage then combines the foreign
peptides with an MHC class 2 molecule that
gets “displayed” on the surface of the
macrophage cell.
Helper T cells that have a T cell receptor
proteins that match (bind to) the displayed
antigen get activated.
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Helper T cells are the central regulatory
element in humoral immunity. They are also
the primary target of the AIDS virus.
More B Cell Activation
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The activated helper T cells then divide (proliferate).
They also activate B cells.
The inactive B cells start the activation process by
binding to an antigen with their IgM molecules,
engulfing it and processing it. The antigen peptides
are displayed on MHC 2 molecules on the cell surface,
just like the macrophages do.
Activated helper T cells interact with B cells that have
the same antigen displayed on their surface: the TCR
on the helper T cell binds to the antigen displayed on
MHC2 on the surface of the B cell.
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The helper T cells secrete cytokines, which stimulate
the B cells to proliferate and differentiate into plasma
cells, which secrete antibodies.
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the CD4 protein on the surface of the helper T cell is also
necessary for this interaction.
this is clonal selection: only those B cells with usable
antibodies are selected to proliferate.
Some descendants of the activated B cell become
plasma cells, while others become memory cells.
Memory cells stay present in the blood for long periods
of time. The next time their specific antigen appears,
the memory cells quickly start proliferating and
differentiating into plasma cells. This provides a rapid
response to a second appearance of the antigen.
Cellular Immunity
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Cytotoxic (or killer) T cells are the main actor in
cellular immunity. Cytotoxic cells (also called
CD8 cells) kill cells that are infected with
viruses or otherwise contain foreign proteins.
Cytotoxic T cells are activated in the same way
that helper T cells are.
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Macrophages eat things in the blood, digest them
to peptide fragments, and display the fragments
on their surfaces bound to MHC class 1
molecules (as opposed to class 2 for helper T
cells).
Inactive cytotoxic T cells have a specific T cell
receptor protein on their surface. If their TCR
binds to the peptide displayed on a macrophage,
the cytotoxic T cell is activated.
the CD8 preotein on the T cell surface facilitates
this interaction.
All cells digest proteins in their lysosomes.
Some of the resulting peptide fragments are
displayed in MHC 1 proteins on the surface of
the cell. Thus, every cell displays a summary
of the proteins inside it on its surface. Mostly
these are acceptable proteins, but if the cell
has been infected by a virus, foreign viral
protein fragments will be displayed.
If the TCR on an activated cytotoxic T cell
binds to a peptide fragment displayed on the
surface of a cell, the T cell kills it by secreting
perforin proteins that punch holes in the cell’s
membrane.
Self vs. Non-self
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If every cellular protein is displayed on the
cell’s surface, and macrophages eat
everything non-selectively, why don’t we
have an immune response against normal
cellular proteins?
The phenomenon of not attacking “self”
proteins is called “tolerance”, and it is the
result of clonal deletion.
Around the time of birth, all the V-D-J joining
involved in making T cell receptors and IgM
molecules has already occurred. Any T cell
(helper or cytotoxic) that binds to an antigen
present in the body at that time is killed
(apoptosis). Thus, “self” is defined as any
molecule circulating in the blood around the
time of birth.
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Clonal deletion occurs in the thymus.
since B cells only become activated by helper
T cells, clonal deletion of T cells ensures that
B cells that might produce antibodies against
self molecules never get activated.
Autoimmune diseases are the result of self
proteins being incorrectly recognized as
non-self.
Immunological Memory
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The first time a new antigen is seen,
the response is slow and relatively
weak.
However, a second appearance of that
antigen gives a rapid, strong reaction.
This is the result of memory cells
formed during the first exposure. The
memory cells have already proliferated
and they quickly start to secrete
antibodies.
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Note that the secondary response is
not general: it is only to the specific
antigen that caused the primary
response.
This is the basis of vaccinations:
introducing a small amount of killed or
weakened virus into the body,
sometimes several times. The body
develops a weak immune response,
but builds up memory cells. When the
pathogen tries to infect the body, a
strong immune response is generated.
Cellular immunity has a similar system
of memory cells.
Rh Incompatibility
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The Rhesus factor is an antigen on the surface of red blood
cells. It was originally found in rhesus monkeys. About 85%
of people have the Rh antigen (Rh+) and 15% lack it (Rh-).
The Rh+ allele is dominant, so heterozygotes are Rh+.
Because the + allele is dominant, it is possible for an Rhmother to have an Rh+ baby.
In general, the fetus’s blood is separated from the mother’s:
there is a barrier against blood mixing at the placenta.
However, during childbirth some of the fetus’s blood often
enters the mother’s body. This causes her immune system
to develop antibodies against the Rh factor.
Since antibodies do cross the placental barrier, a
subsequent Rh+ fetus will have its blood cells attacked by
the mother’s anti-Rh antibodies. This leads to “hemolytic
disease of the newborn”, which used to be a major cause of
stillbirth.
However, these days Rh- mothers are given anti-Rh
antiserum shortly after they give birth to an Rh+ baby. This
antiserum removes any Rh+ blood cells from the mother’s
blood, and so she does not develop an immune response
against Rh.
Severe Combined Immunodeficiency (SCID)
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This disease involves a completely non-functional
immune system due to a lack of T cells.
The “boy in the bubble”. Normally SCID is lethal
within the first year of life. One child was
immediately put into a sterile environment and kept
there until age 12. At this point he was given an
experimental treatment (at his own request).
Unfortunately it failed and he died.
The disease is caused by several different mutant
genes, including those involved with DNA splicing,
interleukins (which signal between macrophages, T
cells, and B cells), the CD4 and CD8 surface
proteins, and others.
Basic treatment: transplant of bone marrow (which
contains blood stem cells) from someone who has
similar tissue antigens.
One form, X-linked SCID, is due to the lack of an
inerleukin receptor. Attempts to cure it by
transfecting a normal version of the gene into blood
stem cells has been fairly successful. However, 2
patients developed leukemia, presumably due to the
insertion of the transfected gene into an oncogene;
gene therapy trial have been suspended.
Another form of SCID is caused by the absence of
adenosine deaminase. This results in the
accumulation of dATP; lymphoid stem cells are very
sensitive to this and die.
Autoimmune Diseases
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A wide spectrum of diseases: rheumatoid arthritis, Crohn’s disease, type 1
diabetes, lupus, and many others.
Normally, immune system cells that react with self antigens are deleted
early in life.
One cause is sequestered antigens: body proteins that did not reach
thymus during the critical period of clonal deletion around birth. Eye lens
crystallin is not generally exposed to the blood, but eye injuries sometimes
allow contact and the development of anti-crystallin antibodies. This can
result in blindness.
Another possible cause is clones of self-reactive immune cells that escape
clonal deletion due to chance events.
A third possible cause is cross reactive antigens. A pathogen carries an
antigen that is similar to a normal body protein. As the body mounts an
immune response against the pathogen, the degree of similarity causes
cross reaction with the body proteins. An example is scarlet fever, which is
caused by a Streptococcus infection. Antibodies produced against these
bacteria often cross react with a protein found on heart valve cells. The
result is heart damage.
No good treatment exists. The main treatments attempt to relieve the
symptoms.
Hypersensitive States
• The body over-reacts to the presence of an antigen.
• There are several types.
– Anaphylactic reaction is a very rapid response that comes from
mast cells releasing large amounts of histamine as a result of
binding the antigen to IgE molecules. Histamine causes the
blood vessels to leak, causing allergy symptoms such as runny
nose and weepy eyes, as well as bronchoconstriction (as in
asthma).
– Some external antigens, especially certain small molecules,
accumulate of the surface of cells. The body’s immune
response can kill the cells.
– accumulation of antibody-antigen complexes in body tissues
can lead to inflammation and tissue damage.
– Contact dermatitis, such as is found in poison ivy rashes, is due
to T cell memory cells,