Immunology

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Immunology

• Chapter 7
• Organization and Expression of Immunoglobulin
  Genes
• Dr. Capers

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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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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