I.
DEFINITION
Immunoglobulin (Ig)
Immunoglobulins are
glycoprotein molecules that are produced by plasma cells in response to an
immunogen and which function as antibodies. The immunoglobulins derive their
name from the finding that they migrate with globular proteins when
antibody-containing serum is placed in an electrical field (Figure 1).
II. GENERAL
FUNCTIONS OF IMMUNOGLOBULINS
A. Antigen
binding
Immunoglobulins bind specifically to one or a few closely related antigens.
Each immunoglobulin actually binds to a specific antigenic determinant. Antigen
binding by antibodies is the primary function of antibodies and can result in
protection of the host. The valency of antibody refers to the number of
antigenic determinants that an individual antibody molecule can bind. The
valency of all antibodies is at least two and in some instances more.
B. Effector
Functions
Frequently the binding of an antibody to an antigen has no direct biological
effect. Rather, the significant biological effects are a consequence of
secondary "effector functions" of antibodies. The immunoglobulins
mediate a variety of these effector functions. Usually the ability to carry out
a particular effector function requires that the antibody bind to its antigen.
Not every immunoglobulin will mediate all effector functions. Such effector
functions include:
1. Fixation of
complement -
This results in lysis of cells and release of biologically active molecules
(see chapter two)
2. Binding to
various cell types - Phagocytic cells, lymphocytes, platelets, mast cells, and
basophils have receptors that bind immunoglobulins. This binding can activate
the cells to perform some function. Some immunoglobulins also bind to receptors
on placental trophoblasts, which results in transfer of the immunoglobulin
across the placenta. As a result, the transferred maternal antibodies provide
immunity to the fetus and newborn
III. BASIC
STRUCTURE OF IMMUNOGLOBULINS
The basic structure of
the immunoglobulins is illustrated in figure 2. Although different
immunoglobulins can differ structurally, they all are built from the same basic
units.
A. Heavy and Light
Chains
All immunoglobulins
have a four chain structure as their basic unit. They are composed of two
identical light chains (23kD) and two identical heavy chains (50-70kD)
B. Disulfide bonds
1. Inter-chain
disulfide bonds - The heavy and light chains and the two heavy chains are held
together by inter-chain disulfide bonds and by non-covalent interactions The
number of inter-chain disulfide bonds varies among different immunoglobulin
molecules.
2. Intra-chain
disulfide binds - Within each of the polypeptide chains there are also intra-chain
disulfide bonds.
C. Variable (V) and
Constant (C) Regions
When the amino acid
sequences of many different heavy chains and light chains were compared, it
became clear that both the heavy and light chain could be divided into two
regions based on variability in the amino acid sequences. These are the:
1. Light Chain - VL (110 amino acids)
and CL (110 amino acids)
2. Heavy
Chain -
VH (110 amino acids) and CH (330-440 amino acids)
D. Hinge Region
This is the region at
which the arms of the antibody molecule forms a Y. It is called the hinge
region because there is some flexibility in the molecule at this point.
E. Domains
Three dimensional
images of the immunoglobulin molecule show that it is not straight as depicted
in figure 2A. Rather, it is folded into globular regions each of which contains
an intra-chain disulfide bond (figure 2B-D). These regions are called domains.
1. Light Chain
Domains -
VL and CL
2. Heavy Chain
Domains -
VH, CH1 - CH3 (or CH4)
F. Oligosaccharides
Carbohydrates are
attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates
may also be attached at other locations.
IV. STRUCTURE
OF THE VARIABLE REGION
A. Hypervariable
(HVR) or complementarity determining regions (CDR)
Comparisons of the
amino acid sequences of the variable regions of immunoglobulins show that most
of the variability resides in three regions called the hypervariable regions or
the complementarity determining regions as illustrated in figure 3. Antibodies
with different specificities (i.e. different combining sites) have different complementarity
determining regions while antibodies of the exact same specificity have
identical complementarity determining regions (i.e. CDR is the antibody
combining site). Complementarity determining regions are found in both the H
and the L chains.
B. Framework
regions
The regions between
the complementarity determining regions in the variable region are called the
framework regions (figure 3). Based on similarities and differences in the
framework regions the immunoglobulin heavy and light chain variable regions can
be divided into groups and subgroups. These represent the products of different
variable region genes.
V. IMMUNOGLOBULIN
FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS
Immunoglobulin
fragments produced by proteolytic digestion have proven very useful in
elucidating structure/function relationships in immunoglobulins.
A. Fab
Digestion with papain
breaks the immunoglobulin molecule in the hinge region before the H-H
inter-chain disulfide bond Figure 4. This results in the formation of two
identical fragments that contain the light chain and the VH and CH1 domains of
the heavy chain.
Antigen binding - These fragments were called the Fab fragments because they
contained the antigen binding sites of the antibody. Each Fab fragment is
monovalent whereas the original molecule was divalent. The combining site of
the antibody is created by both VH and VL. An antibody is able to bind a
particular antigenic determinant because it has a particular combination of VH
and VL. Different combinations of a VH and VL result in antibodies that can
bind a different antigenic determinants.
B. Fc
Digestion with papain
also produces a fragment that contains the remainder of the two heavy chains
each containing a CH2 and CH3 domain. This fragment was called Fc because it was
easily crystallized.
Effector functions -
The effector functions of immunoglobulins are mediated by this part of the
molecule. Different functions are mediated by the different domains in this
fragment (figure 5). Normally the ability of an antibody to carry out an
effector function requires the prior binding of an antigen; however, there are
exceptions to this rule.
C. F(ab')2
Treatment of
immunoglobulins with pepsin results in cleavage of the heavy chain after the
H-H inter-chain disulfide bonds resulting in a fragment that contains both
antigen binding sites (figure 6). This fragment was called F(ab')2 because it
is divalent. The Fc region of the molecule is digested into small peptides by
pepsin. The F(ab')2 binds antigen but it does not mediate the effector
functions of antibodies.
VI. HUMAN
IMMUNOGLOBULIN CLASSES, SUBCLASSES, TYPES AND SUBTYPES
A. Immunoglobulin
classes
The immunoglobulins
can be divided into five different classes, based on differences in the amino
acid sequences in the constant region of the heavy chains. All immunoglobulins
within a given class will have very similar heavy chain constant regions. These
differences can be detected by sequence studies or more commonly by serological
means (i.e. by the use of antibodies directed to these differences).
1. IgG - Gamma heavy
chains
2. IgM - Mu heavy chains
3. IgA - Alpha heavy chains
4. IgD - Delta heavy chains
5. IgE - Epsilon heavy chains
B. Immunoglobulin
Subclasses
The classes of
immunoglobulins can de divided into subclasses based on small differences in
the amino acid sequences in the constant region of the heavy chains. All
immunoglobulins within a subclass will have very similar heavy chain constant
region amino acid sequences. Again these differences are most commonly detected
by serological means.
1. IgG Subclasses
a) IgG1 - Gamma 1
heavy chains
b) IgG2 - Gamma 2 heavy chains
c) IgG3 - Gamma 3 heavy chains
d) IgG4 - Gamma 4 heavy chains
2. IgA Subclasses
a) IgA1 - Alpha 1
heavy chains
b) IgA2 - Alpha 2 heavy chains
C. Immunoglobulin
Types
Immunoglobulins can
also be classified by the type of light chain that they have. Light chain types
are based on differences in the amino acid sequence in the constant region of
the light chain. These differences are detected by serological means.
1. Kappa light
chains
2. Lambda light chains
D. Immunoglobulin
Subtypes
The light chains can also be divided into subtypes based on differences in the
amino acid sequences in the constant region of the light chain.
1. Lambda subtypes
a) Lambda 1
b) Lambda 2
c) Lambda 3
d) Lambda 4
E. Nomenclature
Immunoglobulins are
named based on the class, or subclass of the heavy chain and type or subtype of
light chain. Unless it is stated precisely, you should assume that all
subclass, types and subtypes are present. IgG means that all subclasses and
types are present.
F. Heterogeneity
Immunoglobulins
considered as a population of molecules are normally very heterogeneous because
they are composed of different classes and subclasses each of which has
different types and subtypes of light chains. In addition, different immunoglobulin
molecules can have different antigen binding properties because of different VH
and VL regions.
VII. STRUCTURE AND
SOME PROPERTIES OF IG CLASSES AND SUBCLASSES
A. IgG
1. Structure
The structures of the IgG subclasses are presented in figure 7. All IgG's are
monomers (7S immunoglobulin). The subclasses differ in the number of disulfide
bonds and length of the hinge region.
2. Properties
IgG is the most
versatile immunoglobulin because it is capable of carrying out all of the
functions of immunoglobulin molecules.
a) IgG is the major Ig
in serum - 75% of serum Ig is IgG
b) IgG is the major Ig in extra vascular spaces
c) Placental transfer - IgG is the only class of Ig that crosses the placenta.
Transfer is mediated by a receptor on placental cells for the Fc region of IgG.
Not all subclasses cross equally well; IgG2 does not cross well.
d) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix
complement
e) Binding to cells - Macrophages, monocytes, PMNs and some lymphocytes have Fc
receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2
and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc
receptors on PMNs, monocytes and macrophages is that the cell can now
internalize the antigen better. The antibody has prepared the antigen for
eating by the phagocytic cells. The term opsonin is used to describe substances
that enhance phagocytosis. IgG is a good opsonin. Binding of IgG to Fc
receptors on other types of cells results in the activation of other functions.
B. IgM
1. Structure
The structure of IgM is presented in figure 8. IgM normally exists as a
pentamer (19S immunoglobulin) but it can also exist as a monomer. In the
pentameric form all heavy chains are identical and all light chains are
identical. Thus, the valence is theoretically 10. IgM has an extra domain on
the mu chain (CH4) and it has another protein covalently bound via a S-S bond
called the J chain. This chain functions in polymerization of the molecule into
a pentamer.
2. Properties
a) IgM is the third
most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a
virgin B cells when it is stimulated by antigen.
c) As a consequence of its pentameric structure, IgM is a good complement
fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of
microorganisms.
d) As a consequence of its structure, IgM is also a good agglutinating Ig .
Thus, IgM antibodies are very good in clumping microorganisms for eventual
elimination from the body.
e) IgM binds to some cells via Fc receptors.
f) B cell surface Ig
Surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino
acids at the C-terminus to anchor it into the membrane (figure 9). Cell surface
IgM functions as a receptor for antigen on B cells. Surface IgM is
noncovalently associated with two additional proteins in the membrane of the B
cell called Ig-alpha and Ig-beta as indicated in figure 10. These additional
proteins act as signal transducing molecules since the cytoplasmic tail of the
Ig molecule itself is too short to transduce a signal. Contact between surface
immunoglobulin and an antigen is required before a signal can be transduced by
the Ig-alpha and Ig-beta chains. In the case of T-independent antigens, contact
between the antigen and surface immunoglobulin is sufficient to activate B
cells to differentiate into antibody secreting plasma cells. However, for
T-dependent antigens, a second signal provided by helper T cells is required
before B cells are activated.
C. IgA
1. Structure
Serum IgA is a monomer but IgA found in secretions is a dimer as presented in
Figure 11. When IgA exits as a dimer, a J chain is associated with it.
When IgA is found in secretions is also has another protein associated with it
called the secretory piece or T piece; sIgA is sometimes referred to as 11S
immunoglobulin. Unlike the remainder of the IgA which is made in the plasma
cell, the secretory piece is made in epithelial cells and is added to the IgA
as it passes into the secretions (Figure 12). The secretory piece helps IgA to
be transported across mucosa and also protects it from degradation in the
secretions.
2. Properties
a) IgA is the 2nd most
common serum Ig.
b) IgA is the major class of Ig in secretions - tears, saliva, colostrum,
mucus. Since it is found in secretions secretory IgA is important in local
(mucosal) immunity.
c) Normally IgA does not fix complement, unless aggregated.
d) IgA can binding to some cells - PMN's and some lymphocytes.
D. IgD
1. Structure
The structure of IgD is presented in the Figure 13. IgD exists only as a
monomer.
2. Properties
a) IgD is found in low
levels in serum; its role in serum uncertain.
b) IgD is primarily found on B cell surfaces where it functions as a receptor
for antigen. IgD on the surface of B cells has extra amino acids at C-terminal
end for anchoring to the membrane. It also associates with the Ig-alpha and
Ig-beta chains.
c) IgD does not bind complement.
E. IgE
1. Structure
The structure of IgE is presented in Figure 14. IgE exists as a monomer and has
an extra domain in the constant region.
2. Properties
a) IgE is the least
common serum Ig since it binds very tightly to Fc receptors on basophils and
mast cells even before interacting with antigen.
b) Involved in allergic reactions - As a consequence of its binding to
basophils an mast cells, IgE is involved in allergic reactions. Binding of the
allergen to the IgE on the cells results in the release of various
pharmacological mediators that result in allergic symptoms.
c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels
rise in parasitic diseases, measuring IgE levels is helpful in diagnosing
parasitic infections. Eosinophils have Fc receptors for IgE and binding of
eosinophils to IgE-coated helminths results in killing of the parasite.
d) IgE does not fix complement.
CLINICAL IMPLICATIONS OF HUMAN IMMUNOGLOBULIN CLASSES
Adapted from:F.T.
Fischbach in "A Manual of Laboratory Diagnostic Tests," 2nd Ed., J.B.
Lippincott Co., Philadelphia, PA, 1984.
IgG
1. Increases in:
a) Chronic
granulomatous infections
b) Infections of all types
c) Hyperimmunization
d) Liver disease
e) Malnutrition (severe)
f) Dysproteinemia
g) Disease associated with hypersensitivity granulomas, dermatologic disorders,
and IgG myeloma
h) Rheumatoid arthritis
2. Decreases in:
a) Agammaglobulinemia
b) Lymphoid aplasia
c) Selective IgG, IgA deficiency
d) IgA myeloma
e) Bence Jones proteinemia
f) Chronic lymphoblastic leukemia
IgM
1. Increases (in
adults) in:
a) Waldenström's
macroglobulinemia
b) Trypanosomiasis
c) Actinomycosis
d) Carrión's disease (bartonellosis)
e) Malaria
f) Infectious mononucleosis
g) Lupus erythematosus
h) Rheumatoid arthritis
I) Dysgammaglobulinemia (certain cases)
Note: In the newborn, a level of IgM above 20 ng./dl is an indication of in
utero stimulation of the immune system and stimulation by the rubella virus,
the cytomegalovirus, syphilis, or toxoplasmosis.
2. Decreases in:
a) Agammaglobulinemia
b) Lymphoproliferative disorders (certain cases)
c) Lymphoid aplasia
d) IgG and IgA myeloma
e) Dysgammaglobulinemia
f) Chronic lymphoblastic leukemia
IgA
1. Increases in:
a) Wiskott-Aldrich
syndrome
b) Cirrhosis of the liver (most cases)
c) Certain stages of collagen and other autoimmune disorders such as rheumatoid
arthritis and lupus erythematosus
d) Chronic infections not based on immunologic deficiencies
e) IgA myeloma
2. Decreases in:
a) Hereditary ataxia
telangiectasia
b) Immunologic deficiency states (e.g., dysgammaglobulinemia, congenital and
acquired agammaglobulinemia, and hypogammaglobulinemia)
c) Malabsorption syndromes
d) Lymphoid aplasia
e) IgG myeloma
f) Acute lymphoblastic leukemia
g) Chronic lymphoblastic leukemia
IgD
1. Increases in:
a) Chronic infections
b) IgD myelomas
IgE
1. Increases in:
a) Atopic skin
diseases such as eczema
b) Hay fever
c) Asthma
d) Anaphylactic shock
e) IgE-myeloma
2. Decreases in:
a) Congenital
agammaglobulinemia
b) Hypogammaglobulinemia due to faulty metabolism or synthesis of
immunoglobulins
GENETICS OF
IMMUNOGLOBULINS
I. HISTORY
Amino acid sequencing data revealed that a single C region could be associated
with many different V regions. Also, it was shown that a single idiotype could
be associated with different C regions (eg. IgM and IgG). To explain these data
it was suggested that perhaps the two regions of the immunoglobulin molecule
were coded for by separate genes and that the V and C region genes were somehow
joined before an immunoglobulin molecule was made (i.e. there were two genes
for one polypeptide). This was a revolutionary concept but with the advent of
recombinant DNA technology, it has been shown to be the correct. The
immunoglobulin heavy and light chains are coded for by three separate gene
families each one on a separate chromosome - one for the heavy chain and one
for each of the light chain types. Each of these gene families has several V
region genes and one or more C region genes. The V and C regions genes are not
however immediately adjacent to each other.
II. LIGHT CHAIN
GENE FAMILIES
A. Germ line gene
organization
The organization of
the kappa and lambda light chain genes in the germ line or undifferentiated
cells is depicted in Figure 1.
1. Lambda light chains - The lambda gene family
is composed of 4 C region genes, one for each subtype of lamda chain, and
approximately 30 V region genes. Each of the V region genes is composed of two
exons, one (L) that codes for a leader region and the other (V) that codes for
most of the variable region. Upstream of each of the C genes there is and
additional exon called J (joining). The L, V, J and C exons are separated by
introns (intervening non-coding sequences).
2. Kappa light
chains -
The kappa light chain gene family contains only one C region gene, since there
is only one type of kappa light chain. There are many V region genes
(approximately 250) each of which has a leader exon and a V exon. In the κ gene
family there are several J exons located between the V and C genes. All of the
exons are separated by introns.
B. Gene
rearrangement and Expression
As a cell
differentiates into a mature B cell that will make a light chain, there is a
rearrangement of the various genes (exons) and the gene begins to be expressed
as depicted in Figure 2.
As a cell commits to
become a B cell making a light chain, there is a rearrangement of the genes at
the DNA level such that one of the V genes is brought next to one of the J
regions. This occurs by a recombination event which removes the intron between
the V and J regions. The selection of which V gene is used is not totally
random; there is some preference for the use of V genes nearest to the J
regions. However, with time all V genes can be used so that all combinations of
V genes and J regions can be generated.
A consequence of this
DNA rearrangement is that the gene becomes transcriptionally active because a
promoter (P), which is associated with the V gene, is brought close to an
enhancer (E), which is located in the intron between the J and C regions. As
transcription initiates from the promoter a pre-mRNA is made which contains
sequences from the L, V J and C regions as well as sequences for the introns
between L and V and between J and C (See Figure 2). This pre-mRNA is processed
(spliced) in the nucleus and the remaining introns are removed. The resulting
mRNA has the L, V J and C exons contiguous.
The mRNA is translated in the cytoplasm and the leader is removed as the
protein is transported into the lumen of the endoplasmic reticulum. The light
chain is assembled with a heavy chain in the endoplasmic reticulum and the
immunoglobulin is secreted via the normal route of secretory proteins. The
region V region of the mature light chain is coded for by sequences in the V
gene and J region and the C region by sequences in the C gene.
III. HEAVY CHAIN
GENE FAMILY
A. Germ line gene
organization
The organization of
the heavy chain genes is depicted in Figure 3.
In the heavy chain gene family there are many C genes, one for each class and
subclass of immunoglobulin. Each of the C genes is actually composed of several
exons, one for each domain and another for the hinge region. In the heavy chain
gene family there are many V region genes, each composed of a leader and V
exon. In addition to several J exons, the heavy chain gene family also contains
several additional exons called the D (diversity) exons. All of the exons are
separated by introns as depicted in Figure 3.
B. Gene
rearrangements and expression
As a cell differentiates into a mature B cell that will make a heavy chain,
there is a rearrangement of the various genes segments (exons) and the gene
begins to be expressed as depicted in Figures 4 and 5.
As a cell commits to
become a B cell making a heavy chain, there are two rearrangements at the DNA
level. First, one of the D regions is brought next to one of the J regions and
then one of the V genes is brought next to the rearranged DJ region. This
occurs by two recombination events which remove the introns between the V, D
and J regions. As with the light chains the selection of the heavy chain V gene
is not totally random but eventually all of the V genes can be used.
A consequence of these
DNA rearrangements is that the gene becomes transcriptionally active because a
promoter (P), which is associated with the V gene, is brought close to an
enhancer (E), which is located in the intron between the J and Cmu regions. As
transcription initiates from the promoter a pre-mRNA is made which contains sequences
from the L, V, D, J Cmu and Cdelta regions as well as sequences for the introns
between L and V, between J and Cmu, and between Cmu and Cdelta (Figure 4).
The pre-mRNA is
processed (spliced) in the nucleus and the remaining introns, including those
between the exons in the C genes, are removed (See Figure 5). The pre-mRNA can
be processed in two ways, one to bring the VDJ next to the Cmu gene and the
other to bring the VDJ next to the Cdelta gene. The resulting mRNAs have the L,
V, D, J and Cmu or Cdelta exons contiguous and will code for a mu and a delta
chain, respectively.
The mRNAs are
translated in the cytoplasm and the leader is removed as the protein is
transported into the lumen of the endoplasmic reticulum. The heavy chain is
assembled with a light chain in the endoplasmic reticulum and the
immunoglobulin is secreted via the normal route of secretory proteins. The
region V region of the mature heavy chain is coded for by sequences in the V
gene, D region and J region and the C region by sequences in the C gene.
IV. MECHANISM OF
DNA REARRANGEMENTS
Flanking the V, J and
D exons there are unique sequences referred to as recombination signal
sequences (RSS), which function in recombination. Each RSS consists of a
conserved nonamer and a conserved heptamer that are separated by either 12 or
23 base pairs (bp) as illustrated in Figure 6. The 12bp and 23 bp spaces
correspond to one or two turns of the DNA helix.
Recombination only
occurs between a 1 turn and a 2 turn signal. In the case of the λ light chains
there is a 1 turn signal upstream of the J exon and a 2 turn signal downstream
of Vlambda. In the case of the κ light chains there is a 1 turn signal
downstream of the Vkappa gene and a 2 turn signal upstream of the J exon.. In
the case of the heave chains there are 1 turn signals on each side of the D
exon and a 2 turn signal downstream of the V gene and a 2 turn signal upstream
of the J exon. Thus, this ensures that the correct recombination events will
occur.
The recombination
event results in the removal of the introns between V and J in the case of the
light chains or between the V, D, and J in the case of the heavy chains. The
recombination event is catalyzed by two proteins, Rag-1 and Rag-2. Mutations in
the genes for these proteins results in a severe combined immunodeficiency
disease (both T and B cells are deficient), since these proteins and the RSS
are involved in generating both the B and T cell receptors for antigen.
V. ORDER OF GENE
EXPRESSION IN IG GENE FAMILIES
An individual B cell
only produces one type of light chain and one class of heavy chain. (N.B. The
one exception is that a mature B cell can produce both μ and δ heavy chains but
the antibody specificity is the same since the same VDJ region is found on the
μ and δ chains). Since any B cell has both maternal and paternal chromosomes
which code for the immunoglobulin genes there must be some orderly way in which
a cell expresses its immunoglobulin genes so as to ensure that only one type of
light chain and one class of heavy chain is produced.
The order in which the
immunoglobulin genes are expressed in a B cell is depicted in Figure 7 and 8.
Heavy chain (Figure 7)
A cell first attempts to rearrange one of its heavy chain genes; in some cells
the maternal chromosome is selected and in others the paternal chromosome is
selected. If the rearrangement is successful so that a heavy chain is made,
then no further rearrangements occur in the heavy chain genes. If, on the other
hand, the first attempt to rearrange the heavy chain genes is unsuccessful
(i.e. no heavy chain is made), then the cell attempts to rearrange the heavy
chain genes on its other chromosome. If the cell is unsuccessful in rearranging
the heavy chain genes the second time, it is destined to be eliminated.
Kappa light chain (Figure 8)
When a cell successfully rearranges a heavy chain gene, it then begins to
rearrange one of its kappa light chain genes. It is a random event whether the
maternal or paternal kappa light chain genes are selected. If the rearrangement
is unsuccessful (i.e. it does not produce a functional kappa light chain), then
it attempts to rearrange the kappa genes on the other chromosome. If a cell
successfully rearranges a kappa light chain gene, it will be a B cell that
makes an immunoglobulin with a kappa light chain.
Lambda light
chain (Figure
8) - If a cell is unsuccessful in rearranging both of its kappa light chain
genes, it then attempts to make a lambda light chain. It is a random event
whether the maternal or paternal lambda light chain genes are selected. If the
rearrangement is unsuccessful (i.e. it does not produce a functional lambda
light chain), then it attempts to rearrange the lambda genes on the other
chromosome. If a cell successfully rearranges a lambda light chain gene, it will
be a B cell that makes an immunoglobulin with a lambda light chain.
The orderly sequence
of rearrangements in the immunoglobulin gene families explains:
1) Why an individual B cell can only produce one kind of immunoglobulin with
one kind of heavy and one kind of light chain.
2) Why a individual B cell can only make antibodies of one specificity.
3) Why there is allelic exclusion in immunoglobulin allotypes at the level of
an individual immunoglobulin molecule but co-dominant expression of allotypes
in the organism as a whole.
VI. ORIGIN OF
ANTIBODY DIVERSITY
A. Background
Antibody diversity
refers to the sum total of all the possible antibody specificities that an
organism can make. It is estimated that we can make 107 - 108 different
antibody molecules. One of the major questions in immunology has been how can
we make so many different antibody molecules.
Theories which have
attempted to explain the origin of antibody diversity fall into two major
categories.
1. Germ line
theory
This theory states that we have a different V region gene for each possible
antibody we can make.
2. Somatic mutation theory
This theory state that we have only one or a few V region genes and the
diversity is generated by somatic mutations which occur in these genes.
B. Current Concepts
Our current thinking
is that both the germ line and somatic mutation theories have some merit. It is
thought that antibody diversity is generated by the following mechanisms.
1. A large number
of V genes
There are:
a) 30 lambda V genes
b) 300 kappa V genes
c) 1000 heavy chain V genes
2. V-J and V-D-J
joining
The region where the light chain V gene and J region or the heavy chain V gene
and D and J regions come together is in the third hypervariable region. Since
it is random which V and which J or D regions come together, there is a lot of
diversity that can be generated by V-J and V-D-J joining.
3. Junctional
diversity (Inaccuracies
in V-J and V-D and D-J recombination) - (Figure 9)
Recombination between
V-J and V-D-J is not always perfect and additional diversity can arise by
errors that occur in the recombination event that brings the V region next to
the J or D regions or the D region next to the J region. It is estimated that
these inaccuracies can triple the diversity generated by V-J and V-D-J joining.
The diversity generated by this mechanisms is occurring in the third
hypervariable region and thus, is directly affecting the combining site of the
antibody.
4. N region
insertion
At the junction between D and J segments there is often an insertion of a
series of nucleotides which is catalyzed by the enzyme terminal transferase.
Terminal transferase catalyzes the random polymerization of nucleotides into
DNA without the need for a template. This leads to further diversity in the
third hypervariable region.
5. Somatic
Mutation
There is evidence that somatic mutations are occurring in the V gene,
particularly in the place that codes for the second hypervariable region. Thus,
somatic mutation probably contributes to antibody diversity to some extent.
6. Combinatorial
Association
Any individual B cell has the potential to make any one of the possible heavy
chains and any one of the possible light chains. Thus, different combinations
of heavy and light chains within an individual B cell adds further diversity.
7.
Multispecificity
Due to cross reactions between antigenic determinants of similar structure an
antibody can often react with more than one antigenic determinant. This is
termed multispecificity. Multispecificity also contributes to antibody diversity.
An example of how
these mechanisms can generate a great deal of diversity is illustrated below:
|
|
B Cell Receptor
(Immunoglobulin)
|
|
Heavy
|
Kappa
|
|
V gene segments
|
1000
|
300
|
|
D gene segments
|
15
|
-
|
|
J gene segments
|
4
|
4
|
|
N region insertion
|
++
|
-
|
|
Junctional diversity
|
+++
|
+
|
|
Somatic mutation
|
+
|
+
|
|
Combinatorial
association
|
V x D x J
1000 X 15 X 4
|
V x J
300 x 4
|
|
Total
|
6 x 104
|
1.2 x 103
|
|
|
|
|
Combinatorial
association
|
7.2 x 107
|
These calculations do
not take into consideration the contributions of lambda light chains, somatic
mutation junctional diversity, N region insertions or multispecificity.
The process of gene
rearrangement of the heavy and light chains and the combinatorial association
of these chains occurs during B cell development and is independent of antigen.
Clones of B cells expressing all of the possible antibody specificities are
produced during development and antigen simply selects those clones which have
the appropriate receptor. The selected clones are then activated, proliferate
and differentiate into antibody secreting plasma cells.
VII. T CELL RECEPTOR FOR ANTIGEN
T cells also have a
receptor for antigen on their surfaces. This receptor is not an immunoglobulin
molecule but it is composed of two different polypeptide chains which have
constant and variable regions analogous to the immunoglobulins. Diversity in
the T cell receptor is also generated in the same way as described for antibody
diversity (e.g. by VJ and VDJ joining of gene segments and combinatorial
association). However, no somatic mutation has been observed in T cells.
