Download Lambda Gene Family

Immunoglobulin Genetics
Problem…the immune system makes over
one billion different antibody proteins
• In 1950’s: central dogma stated DNA—to RNA—to
protein
• One gene for each protein
• Required millions of genes just for the immune system
• Does not seem possible, but most scientists thought it
might be
• Today we know the human genome is less than 30,000
genes
• So, what is really going on???
B lymphocyte development
B lymphocyte development (2)
Immunoglobulin Genetics
History
• Same C region could associate with many V regions
– IgG Ab with different specificities
• Same V region could associate with many C regions
– Same idiotype on IgG and IgM Ab
• V and C regions coded for by separate genes
Genes for immunoglobulin proteins are
found on different chromosomes
Light Chain Gene Families
Germ line gene organization
Lambda light chain genes; n=30
V1
L
P
L
V2
P
L
Vn
J
1
P
C
1
J
2
E
J
3
C
2
E
C
3
J
4
E
C
4
E
Kappa light chain genes; n=300
L
P
V1
L
P
V2
L
P
Vn
J 1
J 2
J 3
J 4
J 5
C
E
Light Chain Gene Families
Gene rearrangement and expression
L
V 1
P
L
V 2
P
V n
L
P
L
V 1
P
L
V 2 J4
J 5
P
L
V J
DNA
C
Primary transcript
RNA
L V J C
mRNA
Translation
DNA
C
E
E
RNA Processing
C
E
DNA Rearrangement
Transcription
J 1 J 2 J 3 J 4 J 5
RNA
L V J C
Protein
Transport to ER
V J C
Protein
V
C
Heavy Chain Gene Family
Germ line gene organization
Heavy chain genes; Vn=1000, Dn=15
L
P
V1
L
V2
P
L
D1 D2 D3
Vn
Dn
J1 J2 J3 J4 J5
P
C
C
E
C
C
C
C
3
1
2
4
C
C
C
1
2
CH1 H CH2 CH3 CH4
Introns separate exons coding for H chain domains
Heavy Chain Gene Family
Gene rearrangement and expression
L
P
V1
L
V2
P
D1 D2 D3
Vn
L
Dn
C
J1 J2 J3 J4 J5
C
E
P
DJ rearrangement
L
V1
L
V2
L
D1 D2 J4 J5
Vn
C
C
DNA
P
P
P
E
VDJ rearrangement
L
V1
L
P
V2 D2 J4
P
C
J5
C
DNA
E
Transcription
L
V2 D2 J4
C
J5
E
C
Primary transcript
RNA
Heavy Chain Gene Family
Gene rearrangement and expression
L V2 D2 J4 J5
C
L V2 D2 J4 J5
C
Primary transcript
E
C
C
E
RNA Processing
L V DJ C
L V DJ C
An
An
mRNA for C
mRNA for C
Translation
L V DJ C
L V DJ C
C heavy chain
C heavy
chain
Transport to ER
V DJ C
V
C
C heavy chain
V DJ C
V
C
C heavy
chain
Mechanism of variable region
rearrangements
• Each V, D and J segments of DNA are flanked by
special sequences (RSS—recombination signal
sequences) of two sizes
• Single turn and double turn sequences (each turn
of DNA is 10 base pairs long)
• Only single turn can combine with a double turn
sequence
• Joining rule ensures that V segment joins only
with a J segment in the proper order
• Recombinases join segments together
Mechanism of DNA Rearrangements
• Recombination
signal sequences
(RSS)
– Nonmer
– Heptamer
– 1 or 2 turn signals
• Rag-1 and Rag-2
Nonamer
Nonamer
CACAGTG 23 bp
ACAAAAACC
GGTTTTTGT
12 bp
CACTGTG
23 bp
TGTTTTTGG
CCAAAAACA
12 bp
GTGACAC
Heptamer
GTGTCAC
Two turn
RSS
2
J
2
One turn
RSS
1
Kappa light chains
Lambda light chains
L V
Heptamer
L V
C
J
1
1
Heavy chains
L V
J
D
2
1 1
2
C
2
C
Order of Ig Gene Expression
Progenitor B cell
1st
Heavy chain gene
+
2nd
VDJ recombination
No
Productive rearrangement ?
-
Apoptosis
Yes
Transcribe/translate
 () Heavy chain
Maternal
 or
Pre B cell

Paternal
Order of Ig Gene Expression


or
Pre B cell
Light chain gene
+

VJ recombination
1st - 
2nd - 
3rd - 
4 th
No
Productive rearrangement ?
4th 
-
Apoptosis
Yes
Transcribe/translate
Light chain
+

Mature B cell
Order of Ig Gene Expression
Explains:
• Why an individual B cell only produces one
kind of Ig with one kind of heavy and one
kind of light chain
• Why an individual B cell makes only makes
antibodies of one specificity
• Why there is allelic exclusion
Origin of Antibody Diversity
History
• Definition – Diversity (repitoire) is the total
of all the Ab specificities that an organism
is capable of expressing
• Theories
– Germ line theory
– Somatic mutation theory
Germ-line vs somatic-variation
theories
• Germ-line: stated that each antibody had its own
gene….nothing special, but required billions of
genes to account for numbers of antibodies
• Somatic-variation: some mutation and
recombination created vast number of genes for
antibody formation
• This introduced a new concept: targeted mutation
or recombination of DNA: is it possible??
• Paradox: how could stability be maintained in C
region and diversity exist in V region?
Dreyer and Bennett
• In 1965 proposed radical theory to account for
diversity of antibodies
• Each antibody was coded for by two separate genes
• One for the variable region
• One for the constant region
• Combined at the DNA level and expressed single
mRNA
• Suggested 1000’s of variable region genes and only
one constant region gene
• Close, but no cigar
• Most biomedical scientists did not like this idea and
rejected it
Tonegawa’s demonstration
• 1976—used restriction enzymes and DNA probes to show that germ
cell DNA contained several smaller DNA segments compared to
DNA taken from developed lymphocytes (myeloma cells)
conclusion
• Susumu Tonegawa "Nobel prize winner"
discovered that antibody genes can move
and rearrange themselves within the
genome of a differentiating B cell. The
same applies for TCR.
• Many different V region genes can be
linked up to a single constant "C" region
gene.
Origin of Antibody Diversity
Current concepts
• Multiple V genes
• V-J and V-D-J joining
• Junctional diversity
• N region insertions
– Amino acid
sequences not
encoded in the germ
line
V gene
D region
V gene
D region
CCCCCC
T GG
CCCCCC
T GG
C C C T GG
Pro
C C C C GG
Trp
Pro
V gene
D region
CCCCCC
T GG
CCCCCG
Pro
Pro
Arg
Junctional and Insertional
Diversity
• The absence of precision in joining during
DNA rearrangement leads to deletions or
changes of amino acids "junctional
diversity" that affect Fab region as it occurs
in parts of the hypervariable region.
• Small sets of nucleotides may be inserted
"insertional diversity" at the V-D and D-J
junctions. It is mediated by the enzyme
terminal deoxynucleotidyltransferase "TdT".
The additional diversity generated is
termed N region diversity.
P and N region nucleotide alteration adds to
diversity of V region
• During recombination some nucleotide bases are cut from
or add to the coding regions (p nucleotides)
• Up to 15 or so randomly inserted nucleotide bases are added
at the cut sites of the V, D and J regions (n nucleotides_
• TdT (terminal deoxynucleotidyl transferase) a unique
enzyme found only in lymphocytes
• Since these bases are random, the amino acid sequence
generated by these bases will also be random
Generation of N region insertions
D
J
Break DNA
J
D
TdT adds nucleotides
-
D GTCAATG
J
Ligate
-
-
D GTCAATG J
N region insertion
+
Intron
Origin of Antibody Diversity
Current concepts
• Multiple V genes
• V-J and V-D-J joining
• Junctional diversity
• N region insertions
• Somatic mutation
• Combinatorial association
• Multispecificity
Somatic hypermutation adds even
more variability
• B cell multiplication introduces additional
opportunities for alterations to rearranged
VJ or VDJ segments
• These regions are extremely susceptible to
mutation compared to “regular” DNA,
about one base in 600 is altered per two
generations of dividing (expanding)
lymphocyte population
N.B
All these mechanisms contribute to the
formation of a huge library. Or repertoire of B
lymphocytes that contain all the specificities
required to deal with the multitude of diverse
epitopes that antibodies could encounter. The
number of total Ig specificities that can be
generated in an individual are on the order of
1015 which is increased even more by somatic
hypermutation.
Regulation of Ig Gene Expression
• Each B cell uses only one set of VDJ genes and one type of
light chain. As a result, a single B cell produces an Ig of
only one antigenic specificity.
• Ig chains are coded for by only one set of genes, either from
the maternal or the paternal chromosome, e.g. H chain may
be coded for by genes on the paternal chromosome and the
L chain " or " by genes on the maternal chromosome.
This phenomenon of using genes from only one parental
chromosome is known as allelic exclusion.
• If the process of rearrangement fails in one chromosome,
the second parental chromosome will continue
rearrangement.
• Gene exclusion ensures that every B cell and the Ab it
synthesizes are monospecific e.g. specific for one epitope.
Generation of Diversity
B cell receptor
(Immunoglobulin)
Heavy
Light
V gene segments
1000
300
D gene segments
15
-
J gene segments
4
4
N region insertion
++
-
Junctional diversity
+++
+
Somatic mutation
+
VxDxJ
1000 x 15 x 4
Total
6 x 104
Combinatorial association
• Ag independent process
+
VxJ
300 x 4
1.2 x 103
7.2 x 107
• Clonal selection
ANTIBODY GENE REARRANGEMENTS
CLASS SWITCHING:
Selection of CH Genes
Mechanism Unclear, but Involves
Switch Region
Switch Recombinase
Isotype Specific Cytokine Signals
Copyright © 2007 by W. H. Freeman and Company
Class or Isotype Switching
• One B cell makes Ab of a single specificity that is fixed by
the nature of VJ "L chain" and VDJ "H chain"
rearrangements.
• These rearrangements occur in the absence of Ag in the
early stages of B cell differentiation.
• B cells can synthesize IgM and IgD with the same
antigenic specificity.
• An individual B cell have the ability to switch to make a
different class of Ab... such as IgG, IgE, IgA. This
phenomenon is known as class or isotypic switching.
• Class switching changes the effector function of the B cell
but does not change the cell's antigenic specificity.
Class Switching among Constant-Region Genes
• At the 5` end of every H chain C region "CH"
gene apart from C delta , is a stretch of repeating
base sequences called a switch "S" region.
• This S region permits any of the CH genes "other
than C delta" to associate with the VDJ unit.
• Under the stimulating influence of Ag & T cellderived cytokines, a B cell with a VDJ unit linked
to Cu and C delta further rearranges its DNA to
link the VDJ to an S region in front of another CH
region gene " γ1 in previous fig".
• Cytokines play a key role in CH gene selection
during isotype switching.
• e.g. in the presence of IFN- γ, B cell can
switches to IgG2 synthesis.
• In the presence of IL-4, a B cell can switches to
IgG4 or IgE synthesis respectively.
• Each cytokine is thought to activate an enzyme
known as a "switch recombinase" to recognize
DNA coding for specific C regions.
Understanding of immunoglobulin
structure and formation has opened
up a new world of possibilities
• Monoclonal antibodies
• Engineering mice with human immune
systems
• Generating chimeric and hybrid antibodies for
clinical use
• Abzymes: antibodies with enzyme capability