Immunology Chapter 8

Organization of Immunoglobulin Genes

1.) light chain

• κ light chain has single C gene

• λ light chain has 3 to 6 exons

2.) heavy chains

• VH - DH - JH

• D = diversity region

• each isotope of heavy chain encoded by separate C gene

3.) location

• arranged in 3 clusters (κ, λ, and heavy chains)

• each cluster located on different chromosome

4.) arrangement

• look at figure 5.5

Gene Rearrangement (somatic recombination)

• location:

  • immunoglobulin gene occurs in lymphocytes

  • DNA recombination occurs in germ-line cells (meiosis)

• order of rearrangement:

  • occurs in first heavy chains

    • D & J = gene rearrangement

    • DJ → B & T cells

    • V + DJ = VDJ (B cells)

    • transcribe and splicing RNA = VDJC

  • after VDJ rearrangement in heavy chain, light chain begins

    • κ light chain rearrangement → λ light chain rearrangement

    • V + J = VJ

    • transcribe and splicing RNA = VJC

• mechanism for gene rearrangement (non-homologous recombination):

  1.) RSS

  2.) follows 12/23 rule

  3.) requires lymphocyte specific recombinase

  • proteins encoded by RAG1 & RAG2 forms a dimer

  • dimer is lymphocyte specific recombinase

  • characteristic of lymphocyte specific recombinase:

    • active in B & T cells

    • active in immature lymphocytes

    • recognizes RSS and enforce 23/12 rule

      • NBD (nonomer binding domain) binds to nonmers on RSS

  4.) looping out method

  5.) inversion method

Antibody Diversity

1.) Recombinational diversity

• multiply V, D, and J together to determine possible rearrangements

2.) Combinational diversity

• multiply light and heavy chains together

3.) Junctional diversity

• creates frameshift mutation, stop codons, or nonsense codon

• result in formation of nonfunctional proteins

• AKA non-productive rearrangement

• 3 mechanisms:

  • imprecise DNA rearrangment

    • Artemis cleaves DNA at different places

    • occurs in absence of nonsense or stop codon

    • result = increase in diversity of antibodies

  • N-region diversification

    • look at slide

  • deletion of nucleotides

    • after hairpin open → DNA repair enzyme begins removing nucleotides

    • this occurs at the same time when TdT adds nucleotides and continues until complementary sequences pair up

4.) Secondary diversification

• primary IgM → somatic hypermutation → isotope (class) switching → gene conversion

Expression of Cμ (IgM) and Cδ (IgD)

• In naive B cells, transcription → primary RNA transcript → RNA splicing → makes mRNA

  • transcript splicing:

    • Cδ removed and leaves Cμ → makes IgM

    • Cμ removed and leaves Cδ → makes IgD

  • before activation = removes SC portion of mRNA leaving polyA tail after MC (B cells makes membrane bound antibodies)

  • after activation = B cell → plasma cells = produce more secreted antibodies than membrane bound antibodies (reason why IgM made first during infection)

• In immunoglobulin genes, 2 poly A sites in heavy chain

  • first poly A site = pAs

    • occurs after sequence needed for antibody to be secreted (SC) from cell

  • second poly A site = pAm

    • occurs when downstream of exon encodes transmembrane component of antibody (MC)

    • MC must be present on protein for antibody to be expressed on B cell surface

    • transcription stops at this site

Clonal Selection Hypothesis

• rearrangement occurs once which means each lymphocyte produced can only allow immunoglobulin (antibody) to recognize one epitope

• lymphocyte repertoire = one lymphocyte present to recognize one epitope

  1.) B cell develops in bone marrow

  2.) when body exposed to antigen, B cells and T cells allows immunoglobulin to recognize activated epitope

  3.) B cells and T cells proliferate (make more copies of themselves)

  4.) ones that react with self-antigen are removed

  5.) B cell binds to receptor = B cell activation

  • B cell → plasma cell = secrete antibodies

  • B cell → memory cells

  6.) second exposure to antigen = faster and greater due to presence of memory cells

Secondary Diversification

• only occurs after B cell activation, not T cells

• processes: somatic hypermutation, isotope (class) switching, and gene conversion

• all 3 types involves AID

  • AID = enzyme in activated B cells

  • binds to ssDNA

Somatic Hypermutation

• occurs in activated B cells, germinal centers (AKA dark zone)

• AID = C → U

• UNG removes U = creates abasic site

• mismatch repair = MSH2/6 Poln → mutations in A:T

• REV1 → mutations in C:G

Selection of T cells (affinity maturation):

1.) B cells migrate to antigen in dark zone of germinal center

2.) B cells with high affinity of antigen capture and process presentation by MHCII molecules

3.) B cells that can present antigen to TFH cells will receive survival and mitogenic signal via CD40 & cytokines

• T cell activated → produces CD40L → binds to CD40 on B cell

• T cell produces IL-21

• CD40L and IL-21 = survival signal

4.) B cells that has help from TFH cells can re-enter dark zone to undergo additional mutations (somatic hypermutation and proliferation)

Gene Conversion

• does NOT occur in humans

• APE1 → single stranded nicks

• recombination occurs between nick in V and and another V

• results in insertion of new V into VDJ = creating diversity

Isotope (class) Switching

• occurs after activation by antigen