Organization and Expression of Immunoglobulin Genes

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• Chapter 7
• Organization and Expression of Immunoglobulin
  Genes

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• 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?

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• 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

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• 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

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• 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 its specific for
• Now the B cell can switch from making IgM to IgD
  to IgG, etc
• All with the same variable region

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• Kappa (?) and lamda (?) 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

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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

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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.

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• 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.
  

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• 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.

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• 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.

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• 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 testthe
  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.

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• 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 cells
  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)

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• 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)

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• 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.

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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.

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• 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 re-stimulated 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.

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• 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.

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• BUT, hypermutation in TCRs is not beneficial
  (remember you want them to recognize self---but
  not over reactgtgtautoimmune problems)

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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

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Mechanism of Variable-Region DNA rearrangements

• Catalyzed by enzymes
• V(D)J recombinase
• Proteins mediate V-(D)-J joining
• RAG-1 and RAG-2

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• 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?vAxIMmNByqtM
• 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)

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Allelic Exclusion

• Ensures that the rearranged heavy and light chain
  genes from only 1 chromosome are expressed

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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

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Ab diversity Multiple gene-line segments AND
combination of those segments
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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

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Ab diversity P-addition and N-addition
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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

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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

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µ?d???e?a IgM?IgD?IgG?IgE?IgA
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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

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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

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Antibody Engineering

• Monoclonal Abs used for many clinical reasons
  (anti- tumor 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

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Rearrangement of TCR genes

• Similar to that of Ig
• Rearrangement of a and ? chains
• V, J, and C segments
• Rearrangement of ß and d chains
• V, D, J, and C segments

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• 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 doesnt change to cause self-reactive T cell