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