Transcript Immunoglobulin Genes: Organization and Expression

Immunoglobulin Gene
Organization and Expression
W. Robert Fleischmann, Ph.D.
Department of Urologic Surgery
University of Minnesota Medical School
[email protected]
(612) 626-5034
Objectives
• Understand how the immunoglobulin genes are
organized in the DNA.
• Understand the basis for diversity of
immunoglobulins.
• Understand the mechanisms by which the
variable regions undergo gene rearrangement.
• Understand the mechanism of class switching.
• Understand how immunoglobulin genes are
expressed.
• Understand the medical consequences of the
activity of genetic rearrangement mechanisms.
Patti Palmer, age 7 months, is brought by her
parents to your clinic for testing. For the past 3
months, she has been having repeated bouts of
bacterial sinusitis, otitis media, and lung
infections. Streptococcus pneumoniae has
been cultured from Patti during several of these
infections, despite the fact that she has been
vaccinated with pneumococcal conjugate
vaccine at 2 months, 4 months, and 6 months.
She is currently ill with a lung infection.
What thoughts do you have about Patti?
The physician suspects that Patti has an
immundeficiency.
Blood test results:
Total WBC:
19,100/µl (4,100-10,900/µl)
Differential WBCs:
Neutrophils
82%
(35-80%)
Lymphocytes
15%
(20-50%)
Macrophages
3%
(2-12%)
Eosinophils
0%
(0-7%)
Basophils
0%
(0-2%)
All other blood test results are normal.
What thoughts do you have about Patti?
What additional tests might you wish to run?
General Structure of an
Antibody
Light chains:
Kappa chains are found twice as
frequently in Abs as are lambda chains.
Heavy chains:
These undergo class switching
as the B cell undergoes differentiation.
Light chain
(kappa or lambda)
Heavy chain
(isotypic region:
,  , , or )
The Conundrum
• The human genome contains an estimated
20,000 to 25,000 genes that encode mRNAs
for proteins.
• To protect us, our immune system has the
ability to produce about 1010-1011 different
antibodies.
• If every gene encoded a different antibody,
the genes would encode only 1 millionth of
the antibodies needed.
• How can our immune system produce so
many different antibodies with so few genes?
Features of the Antibody Genes
• Antibodies are composed of heavy and light chains.
• As is seen for most eukaryotic genes, the heavy and
light chains of the immunoglobulin genes are each
composed of segments (exons) that must be joined
together to form the immunoglobulin genes.
• For immunoglobulin genes, the joining of a number of
the exons occurs via a rearrangement of the gene
segments at the level of the DNA, rather than at the
level of the mRNA.
• There are multiple copies of each of the various
segments of the heavy and light chains of the
immunoglobulin genes, with one of each of these
segments becoming sequentially rearranged to form
the heavy and light chain genes.
The Keys to Antibody Diversity
• Antibody diversity is generated during genetic
rearrangement by mixing and matching one of each
of the various gene segments for the heavy and light
chains in a combinatorial manner.
• Antibody diversity is generated by errors incorporated
at the joining sites for the various segments of the
heavy and light chains.
• Antibody diversity is generated by hypermutation in
one of the gene segments (variable regions) of the
heavy and light chains during proliferation of B cells.
• Antibody diversity is generated by mixing and
matching heavy and light chains in a combinatorial
manner.
Overview of
Immunoglobulin
Expression at
Various Stages of
B Cell Maturation
The Immunoglobulin Heavy Chains
• There are two identical immunoglobulin
heavy chains in each antibody.
• Each of the immunoglobulin heavy chain
genes is assembled from V, D, J, and C gene
segments.
• There are multiple C gene segments
(constant regions) that give rise to different
isotypes.
–
–
–
–
–
IgD
IgM
IgG: 4 heavy chains, IgG1, IgG2, IgG3, IgG4
IgE
IgA: 2 heavy chains IgA1, IgA2
Heavy Chain Rearrangement
Heavy Chain Diversity
• 39 V gene segments
• 23 D gene segments
• 6 J gene segments
5,382 combinations of heavy chain VDJ
segments
The Immunoglobulin Light
Chains
• There are two types of immunoglobulin
light chains.
  light chain
  light chain
• Each of the immunoglobulin light chain
genes is assembled by the
rearrangement of V, J, and C gene
segments.
 Light Chain Rearrangement
Light Chain Diversity
  light chain diversity
– 40 V gene segments
– 5 J gene segments
– 1 C gene segment
  light chain diversity
– 30 V gene segments
– 4 J gene segments
– 4 C gene segments
200 combinations of  light chain VJC
segments
480 combinations of  light chain VJC
segments
Antibody Diversity from
Rearrangements
• From heavy chains: 5,382 combinations
• From  and  light chains: 680 combinations
3.6 x 106 Combinations of light and heavy
chains
This is far short of the 1010-1011 different
Antibodies that are postulated to occur.
How Does Rearrangement
Occur?
• Rearrangement occurs between specific
sites on the DNA called recombination
signal sequences (RSSs).
• Rearrangement is catalyzed by two
recombination-activating genes: RAG-1
and RAG-2.
Recombination Signal Sequences
• The specific recognition sequences, called recombination signal
sequences (RSSs), indicate the sites of recombination.
• These signal sequences composed of a 7 bp sequence and a 9 bp
sequence are separated by one turn of the DNA (12 bp spacer) or by
two turns of the DNA (23 bp spacer).
• The RSSs are inverted repeats that allow the DNA to form a stemloop with the RSSs aligning on the stem.
Rearrangement Process
Site of P and N nucleotide
additions
P-Nucleotide And
N-Nucleotide Additions
• Cleavage of the RSSs by RAG
leaves single-stranded regions.
• These single-stranded regions
are copied to form a hairpin loop
by addition of P-nucleotides (P
for palindromic nucleotides).
• Additional N nucleotides (N for
any nucleotide) may be added.
• The sequences are joined.
Consequences of Rearrangement and
P- and N-Nucleotide Addition
• Positive consequence of imprecise joining of Ig gene
segments
– A productive rearrangement occurs if the number of nucleotides
added across the joining region allows the genetic code to be
read in phase.
– This results in the generation of additional diversity.
• Negative consequence of imprecise joining of Ig gene
segments
– A nonproductive rearrangement occurs if the number of
nucleotides added across the joining region causes the genetic
code to be read out of phase (the majority of times).
– This results in an incomplete antibody (run into stop codons)
– The B cell may be able to productively rearrange the
immunoglobulin gene on the other chromosome.
– Otherwise, it will result in the death of the B cell.
Alleleic Exclusion
• Alleleic exclusion occurs when only one of two
alleles is expressed.
• This is the case with immunoglobulin
molecules.
• This ensures that a given B cell will make
antibody molecules with only a single
specificity.
• Antibodies may be made from maternal and
paternal chromosomes:HM:LM; HP:LM; HM:LP;
HP:LP.
Note: If the first allele makes a non-functional
antibody, the second allele will undergo
rearrangement.
Immunoglobulin Molecules
Expressed on Mature B Cells
• Mature (but not activated) B cells initially express IgD
and IgM on their external cell membranes.
– The choice of IgD versus IgM occurs at the level of processing of
mRNA, so a given B cell can both express IgD and IgM.
• As mature B cells are activated to divide and differentiate
by their cognate antigen, they switch from membranebound IgD and IgM to secretory IgM.
– This switch occurs at the level of processing of mRNA
transcripts.
• As they continue to divide and differentiate, they may
undergo additional class switching: IgM => IgG => IgE
=> IgA.
– These switches occur at the level of rearrangements of the DNA.
Expression of Membrane-Bound IgD and IgM
•
•
•
•
An initial pre-mRNA
transcript is produced.
Importantly, the premRNA transcript has
two poly-A sites.
If the second
polyadenylation site is
read, then the mRNA
for membrane-bound
IgM is generated by
splicing.
If the fourth
polyadenylation site is
read, then the mRNA
for membrane-bound
IgD is generated by
alternate splicing.
Structures of Membrane IgM and Secreted IgM
Note the cysteine
for creation of S-S
bonds between IgM
antibodies.
Note the many
hydrophobic
amino acids in
the transmembrane
sequence.
Expression of MembraneBound IgM and Secreted IgM
• The initial premRNA transcript is
synthesized.
• Importantly, the
pre-mRNA
transcripts have
two poly-A sites
within the Cµ gene
segment.
• If the M1, M2
exons are spliced
out, the mRNA for
secreted IgM is
produced.
Class Switching With Activated B
Cell Differentiation And Division
• After activation, B cells switch from
membrane-bound IgM and IgD to secreted
IgM by differential splicing.
• As the activated B cells continue to
differentiate and divide, they class switch to
production of IgG by DNA rearrangement.
• Activated B cells may continue to class switch
to production of IgE or IgA by DNA
rearrangement.
Mechanism of Class Switching
•
•
•
•
With activation of
the B cell, class
switching can occur.
At the level of the
DNA, a looping
event occurs that
cuts out the
constant regions for
IgM and IgD.
This leads to the
production of IgG
mRNAs.
If further looping out
occurs, the mRNAs
for IgE or IgA are
produced.
Cytokine Effects on Class
Switching
Certain cytokines
affect class
switching:
IFN- => IgG2a
IL-4 => IgG1, IgE
IL-5 => IgE
Antibody Diversity by Hypermutation
• After exposure to its cognate
antigen, the mature B cell is
activated to proliferate.
• As the B cell proliferates,
mutations accumulate in the
immunoglobulin gene by a
process called somatic
hypermutation.
• These mutations are
concentrated in the variable
region.
• The mutations give greater
antibody diversity.
• Some of the mutations will
lead to an antibody that
binds the antigen more firmly
(affinity maturation).
• If the mutations lead to less
Ab affinity, the B cell dies.
Review of
Immunoglobulin
Expression at
Various Stages of
B Cell Maturation
Patti Palmer
Lymphocyte stimulation test:
Phytohemaglutinin
136.2
Concanavalin A
47.9
Pokeweed mitogen
49.3
How do you interpret these results?
What additional tests would you wish to run?
134.3
48.8
46.5
Patti Palmer
Antibody levels in the blood:
Serum IgG
385 mg/100 ml
Serum IgA
30 mg/100 ml
Serum IgM
50 mg/100 ml
350-800 mg/100 ml
15-50 mg/100 ml
25-75 mg/100 ml
How do you interpret these results?
What additional tests would you wish to run?
Patti Palmer
IgG subtype test:
Different IgG subtypes were measured,
using ELISA. The level of IgG2 was
below the detectable level. The levels of
IgG1, IgG3, and IgG4 were all in the
normal range.
How do you interpret these results?
Patti Palmer
Patti is diagnosed with IgG2 subclass
deficiency. There is no rearrangement of her
IgG2 heavy chain, perhaps because of a lack of
switch regions for her IgG2 heavy chain.
A principal cause of recurrent pneumococcal
infections is a deficiency in the antibody
response to pneumococcal polysaccharides in
the capsule. IgG2 is the antibody subtype that
best recognizes polysaccharides. Thus, a
deficiency in IgG2 leads to an inability to control
pneumococcal infections.
Patti is begun on intravenous IgG at 3-4 week
intervals.
Diseases Associated With
Expression of Immunoglobulin
Genes
•
•
•
•
•
Burkitt’s Lymphoma
Acute Lymphocytic Leukemia
Acute Myelogenous Leukemia
Chronic Lymphocytic Leukemia
Chronic Myelogenous Leukemia
Diseases Involving
Translocations of
Oncogenes Onto
Immunoglobulin Genes
• Chronic Myelogenous Leukemia:
– Philadelphia chromosome (novel
chromosome 22)
– This results from a reciprocal
translocation of chromosomes 9 and
22.
– This places a proto-oncogene under
the control of the  light chain gene
promoter on chromosome 22.
• Burkitt’s Lymphoma:
– A reciprocal translocation of
chromosomes 8 and 14.
– This places c-myc under the control of
the heavy chain promoter on
chromosome 14.
Medical Uses of Antibodies
• Monoclonal antibodies are used for
treatment of certain diseases.
Anti-Idiotopic Antibody
• The concept is to make an
antibody that mimics an
antigen.
• This is not in use yet.
• It could be used to generate
antibodies against antigens.
– Antigens that are not readily
recognized: a polysaccharide
molecule
– Antigens that are not readily
recognized and toxic: the lipid
A region of bacterial endotoxin.
• The anti-idiotype antigen
could then be used as a
vaccine.
Generation of
Monoclonal Antibodies
• B cells from recently immunized mice
are harvested from spleens and fused
with mouse myeloma cells.
• The myeloma cells have been cloned to
resist a specific toxic drug.
• Only myeloma cells and fused myeloma
cells survive treatment with the toxin.
• The surviving cells are cloned and
tested for production of antibody to the
antigen used to immunize the mice.
• The identified clone is cultured and
harvested for monoclonal antibody.
Production of Humanized
Monoclonal Antibodies
• Harvest mouse embryonic stem cells and
KO the genes for the mouse heavy and
light chains.
• Transfect the KO mouse cell with a human
artificial chromosome that encodes the
human heavy and light chains.
• Inject into a mouse blastocyst to generate
a chimeric mouse with 1 normal mouse
and one KO mouse chromosome for
antibody genes.
• Interbreed progeny mice to obtain a mouse
that is homozygous for the KO mouse
chromosome and the human artificial
chromosome.
• Inoculate mice with antigen as for
generating monoclonal antibody.
Immunize with antigen.
Harvest spleen cells.
Fuse with myeloma cells.