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Chapter 7
Organization and Expression of Immunoglobulin Genes

How does antibody diversity arise?

What causes the difference in amino acid
sequences?

How can different heavy chain constant
regions be associated with the same
variable regions?

In germ-line DNA, multiple gene
segments code portions of single
immunoglobulin heavy or light chain
 During B cell maturation and stimulation, gene
segments are shuffled leaving coding sequence
for only 1 functional heavy chain and light
chain
○ Chromosomal DNA in mature B cells is not the
same as germ-line DNA

Dreyer and Bennett – 1965
 2 separate genes encode single immunoglobulin
heavy or light chain
○ 1 for the variable region
 Proposed there are hundreds or thousands of these
○ 1 for the constant region
 Proposed that there are only single copies of limited classes

Greater complexity was revealed later
 Light chains and heavy chains (separate multi-gene
families) are located on different chromosomes

DNA rearrangement: produces variable
region
○ Happens before the B cell encounters antigen

Later mRNA splicing: produces constant
region
○ Happens after that particular B cell encounters
antigen it’s specific for
○ Now the B cell can switch from making IgM to
IgD to IgG, etc
 All with the same variable region


Kappa (κ) and lamda (λ) light chain segments:
○ L – leader peptide, guides through ER
○ V
VJ segment codes for variable region
○ J
○ C – constant region
Heavy chain
○ L
○ V
VDJ segment codes for variable region
○ D
○ J
○ C
Variable-region gene rearrangements

Variable-region gene rearrangements
occur during B-cell maturation in bone
marrow
○ Heavy-chain variable region genes rearrange first
○ Then light-chain variable region
○ In the end, B cell contains single functional
variable-region DNA sequence
○ Heavy chain rearrangement (“class switching”)
happens after stimulation of B cell
Heavy Chain Rearrangement

The B cell receptor is made up of two kinds
of proteins, the heavy chain (Hc) and the
light chain (Lc)., Each of these proteins is
encoded by genes that are assembled from
gene segments.

Each B cell has 2 chromosome 14s (Mom +
Dad)…but a B cell makes only one kind of
Ab. So the segments on one chromosome
have to be “silenced”.

This works like a card game with 2 players.
It is “winner takes all”..each player tries to
rearrange its card (gene segments) until it
finds a arrangement that works. The first
player to do this wins.

The players in the card game first choose
one each of the possible D and J segments,
and these are joined deleting the DNA
sequences in between.

Then one of the many V segments is
chosen, and this “card” is joined to the DJ
segment, again by deleting the DNA in
between.

Next to the rearranged J segment is a
strong of gene segments (CM, CD, etc) that
code for the various constant regions.

By default, the constant regions for IgM
and IgD are used to make the BCR, simply
because they are first in line.
Next, the rearranged gene segments are
tested. (if the gene segments are not lined
up right, the protein translation machinery
will encounter a stop codon and terminate
protein assembly.
 If the segment passes the test, that
chromosome is used to construct the
winning Hc protein. This heavy chain
protein is then transported to the cell
surface, where it signals to the losing
chromosome that the game is over.


If the heavy chain rearrangement is
productive, the baby B cell proliferates for
a bit, and then the light chain players step
up to the table. The rules of the game are
similar to those of the heavy chain game,
but there is a second test…the completed
heavy chain and light chain proteins must
fit together properly to make a complete
antibody. If this does not occur, the B cell
commits suicide.
To produce antibody the B cell has to be
activated. “Naïve” or “virgin” B cells have
never encountered their antigen.
 Activation of a naïve B cell requires 2
signals: the first is the clustering of the B
cell’s receptors and their associated
signaling molecules. A second signal is
required (co-stimulatory signal) (Note: in
T cell-dependent activation, this second
signal is supplied by a helper T-cell).
 In response to certain antigens, naïve B
cells can also be activated with little or no
T cell help (T-cell independent)


Once B cells have been activated, and have
proliferated to build up their numbers,
they are ready for maturation. Maturation
occurs in 3 steps:
 class switching (where a B cell can change the
class of antibody it produces)
 somatic hypermutation (rearranged genes for
the BCR can undergo mutation and selection that can increase
the affinity of the BCR for the antigen
 career decision (B cell decides whether to
become a plasma or memory cell)


Virgin B cells first produce IgM (default). As the B
cell matures, it has the opportunity to change the
class of Ab to either IgG, IgE or IgA.
The gene segments that code for the constant region
for IgM are next to the constant regions for IgG, IgE
or IgA, switching is easy:
 Cut off the IgM constant region DNA and
paste on one of the other constant regions.
Somatic Hypermutation
Normal overall mutation rate of DNA is
extremely low (~ 1 mutated base /100
million bases). However, the
chromosome area where B cell are encoded
is highly restricted, which means that an
extremely high rate of mutation can occur
(~1 mutated base per 1000 cases).
 This high rate of mutation is called somatic
hypermutation. It occurs after the V,D,
and J segments have been selected, and
usually after class switching.

Somatic hypermutation changes the part of
the rearranged Ab gene that encodes the
antigen binding region of the Ab. Depending
on the mutation, there are three possible
outcomes.
 The affinity of the Ab for the Ag may remain
unchanged, my increase or may decrease
 For maturing B-cells to continue to
proliferate, they must be continually restimulated by binding to their Ag.
Therefore, because those B cells whose BCRs
have mutated to a higher affinity are
stimulated more easily, they proliferate more

frequently.

Because they proliferate more frequently,
the result is that you end up with many
more B cells whose BCRs have high affinity
for their Ag.

BUT, hypermutation in TCRs is not
beneficial (remember you want them to
recognize self---but not over
react>>autoimmune problems)
Mechanism of Variable-Region DNA rearrangements

Recombination signal sequences (RSSs)
○ Between V, D, and J segments
○ Signal for recombination
○ 2 kinds
- 12 base pairs (bp) – 1 turn of DNA
- 23 bp – 2 turns of DNA
- 12 can only join to 23 and vice versa
Mechanism of Variable-Region DNA rearrangements

Catalyzed by enzymes
○ V(D)J recombinase

Proteins mediate V-(D)-J joining
○ RAG-1 and RAG-2

Gene arrangements may be nonproductive
○ Imprecise joining can occur so that reading frame is not complete
○ Estimated that less than 1/9 of early pre-B cells progress to maturity

Gene rearrangement video:
 http://www.youtube.com/watch?v=AxIMmNByqtM

Look at Figure 7-8 – VDJ recombination
○ 1. Recognition of RSS by RAG1/RAG2 enzyme complex
○ 2. One-strand cleavage at junction of coding and signal sequences
○ 3. Formation of V and J hairpins and blunt signal end
○ 4. ligation of blunt signal end to form signal joint
- 2 triangles on each end (RSS) are joined
○ 5. Hairpin cleavage of V and J regions
○ 6. P nucleotide addition (palindromic nucleotide addition – same if read
5’ to 3’ on one strand or the other
○ 7. Ligation of light V and J regions (joining)
○ 8. Exonuclease trimming (in heavy chain)
- Trims edges of V region DNA joints
○ 9. N nucleotide addition (non-templated nucloetides)
○ 10. Ligation and repair
Allelic Exclusion

Ensures that the rearranged heavy and light
chain genes from only 1 chromosome are
expressed
Generation of Antibody Diversity
Multiple germ-line gene segments
 Combinatorial V-(D)-J joining
 Junctional flexibility
 P-region nucleotide addition
 N-region nucleotide addition
 Somatic hypermutation
 Combinatorial association of light and
heavy chains

○ This is mainly in mice and humans – other studied species differ in
development of diversification
Ab diversity – Multiple gene-line segments
AND combination of those segments
Ab diveristy – junctional flexibility

Random joining of V-(D)-J segments
○ Imprecise joining can result in nonproductive
rearrangements
○ However, imprecise joining can result in new
functional rearrangements
Ab diversity – P-addition and N-addition
Ab diversity – somatic hypermutation
Mutation occurs with much higher
frequency in these genes than in other
genes
 Normally happens in germinal centers in
lymphoid tissue

Class Switching
Isotype switching
 After antigenic stimulation of B cell
 VHDHJH until combines with CH gene
segment
 Activation-induced cytidine deaminase
(AID)

 Somatic hypermutation
 Gene conversion
 CLASS-SWITCH recombination

IL-4 also involved
μ→δ→γ→ε→α
IgM→IgD→IgG→IgE→IgA
Ig Gene Transcripts

Processing of immunoglobulin heavy
chain primary transcript can yield several
different mRNAs
○ Explains how single B cell can have secreted and
membrane bound Ab
Regulation of Ig-Gene Transcription

2 major classes of cis regulatory sequences in DNA regulate
 Promoters – promote RNA transcription in specific direction
 Enhancers – help activate transcription
 Gene rearrangement brings the promoter and enhancer closer
together, accelerating transcription
Antibody Engineering



Monoclonal Abs used for
many clinical reasons (antitumor Ab, for instance)
If developed in mice, might
produce immune response
when injected
○ Can be cleared in
which they will not be
efficient
○ Can create allergic
response
Creating chimeric Abs or
humanized Abs are
beneficial
Rearrangement of TCR genes

Similar to that of Ig
 Rearrangement of α and γ chains
○ V, J, and C segments
 Rearrangement of β and δ chains
○ V, D, J, and C segments

Generation of TCR diversity (a lot like Ig)
○ Multiple germ-line gene segments
○ Combinatorial V-(D)-J joining
○ Junctional flexibility
○ P-region nucleotide addition
○ N-region nucleotide addition
○ Combinatorial association of light and heavy chains

However, there is no somatic mutation with
TCR
○ May be to ensure that after thymic selection, the TCR
doesn’t change to cause self-reactive T cell