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Organization and Expression of�Immunoglobulin Genes

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Organization of Immunoglobulin Genes�

  • Numerous V region genes are preceded by Leader or signal sequences (60-90 bp) exons interspersed with introns.
  • Heavy chain contains V (Variable), D (Diversity), J (Joining) and C (Constant) region gene segments.
    • V - D - J – C
  • Light chain contains V, J, and C region gene segments
    • V - J - C
  • Constant region genes are sub-divided into exons encoding domains (CH1,CH2, CH3, CH4)

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Kappa Light Chain Gene Organization and Rearrangement

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Heavy Chain Gene Organization and Rearrangement

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MECHANISM OF IMMUNOGLOBULIN RE-ARRANGEMENT

  • Occurs principally via looping out (excision) of intervening gene sequences followed by ligation of Ig gene segments.
  • Controlled by recombination signal sequences (RSS) located at joining sites.
  • Consist of heptamer/nonamer (7/9) sequences interspersed by 12/23 base pair spacers.
  • Recognized by Recombinases (enzymes with endonuclease and ligase activities).
  • Consists of RAG1,2 proteins (lymphocyte-specific), and nonlymphocyte- specific DNA repair proteins (DNA ligase IV), DNAdependent protein kinase (DNA-PK) and Ku, a protein that associates with DNA-PK
  • Genes encoding recombinases are present in all cell types but are expressed only in lymphoid (B &T) cells.
  • Recombination activating genes 1 and 2 (RAG-1, RAG-2) have been identified which stimulate Ig gene rearrangement. Have endonuclease activity

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Recombination of immunoglobulin variable region genes.

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Recombination between gene segments is required to generate a complete H/L chain

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Antibody Diversity Mechanisms

  • To date, seven means of antibody diversification have been identified in mice and humans:
    • Multiple germ-line gene segments
      • Combinatorial V-(D)-J joining
    • P-region nucleotide addition (P-addition)
    • N-region nucleotide addition (N-addition)
    • Junctional flexibility
    • Somatic hypermutation
    • Combinatorial association of light and heavy chains

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  • Junctional Diversity
    • Imprecise joining
    • N/P region (insertional) diversity occurs in VDJ joining (heavy chain) as well as VJ join of light chain. Arises from addition of up to 20 nucleotides by terminal deoxynucleotidyl transferase (TdT).

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Somatic Hypermutation

    • Occurs randomly after antigenic stimulation and principally in CDR1, CDR2, CDR3 regions (more frequent in CDR3).
    • Introduces point mutations at a higher rate than for normal mammalian genes.
    • Mutation rate of V genes is 1 base pair change per 103 base pairs/cell division; it is 10-7 in other mammalian genes.
    • Can give rise to Ig with different (new) antigen specificities leading to high or low affinity Abs. High affinity B cell clones are selectively expanded (Affinity Maturation).
    • Affinity maturation is associated with isotype switching.

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ISOTYPE SWITCHING

  • Is the conversion of an immunoglobulin from one isotype to another (e.g. IgG to IgE) while retaining the same antigen specificity.
  • Switching is dependent on antigenic stimulation and is induced by cytokines released by helper T cells and requires engagement of CD40L e.g. IL-4 triggers switching from IgM to IgE or IgG4 (humans)
  • Cyokines are thought to alter chromatin structure making switch sites more accessible to recombinases for gene transcription.
  • Involves switch sites located in introns upstream of each CH segment (except Cδ).
  • Class switching occurs usually in activated B cells (including memory cells) and not in naïve B cells and involves heavy chain genes.
  • These cells (you will recall) already have rearranged VDJ genes at the DNA

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T cell receptor

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