Origin of diversity Bibliography. Antibody diversity: one enzyme to rule them all Michel C Nussenzweig & Frederick W Alt Nature Medicine 10;1304-5;2004 Immunobiology Janeway et al 6 th ed 2005 (or 7 th ed. 2008). Clonal selection theory. יצירת מאגר של לימפוציטים בשלים. תא אב יחיד מתמיין
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Antibody diversity: one enzyme to rule them all
Michel C Nussenzweig & Frederick W Alt
Nature Medicine 10;1304-5;2004
Immunobiology Janeway et al 6th ed 2005 (or 7th ed. 2008)
יצירת מאגר של
תא אב יחיד מתמיין
למספר גדול של
אחד מהם ספציפיות
קלון של תאים
סילוק של לימפוציטים
לא בשלים המגיבים
עם אנטיגנים עצמיים
The chance for successful rearrangement is 33%
IgG “Memory” cell
Heavy chain +
IgM + IgD
IL-7R (IL-7Ra and IL-2 gc chain)
Iga and Igb proteins
IL-7 and adhesive interactions between B cells and stromal cells important for proliferation
1. clonal expansion
2. allelic exclusion
3. light chain rearrangement
l5 KO blocks L chain rearrangement
and B cell differentiation
It does not block second H-chain
Sensitive to self
After gene rearrangements and production of a functional molecule, the cell tests whether its specificity is anti-self
Maintenance of tolerance requires the persistence of antigen
because self-antigens are always present but foreign antigens are transient
Before clonal deletion of an anti-self B cell, the cell can attempt receptor editingof the light chain
is still on
So, light chain can use repeated rearrangements and can receptor edit. Repeated rearrangements are to make a functional molecule whereas receptor editing is to avoid clonal deletion of anti-self specific B cells.
Immature B cell “edits” light chain if it binds antigen (gets negative selection signal). This could rescue the cell from negative selection (i.e., death). (needs signal to edit)
Immature T cells continues to rearrange a chain until the cell gets positive selection signal. (will eventually die if it does not receive positive selection in a few days). (needs signal to stop “editing”)
Va are mixed with Vd
This provides for many attempts at a productive rearrangement. Rearrangements
stop when there is positive selection.
diversity in humans
H-L chain associations
RAG 1 & 2 required
In synthetic substrate transfected into cell lines and mice, deletion of any of promoters or enhancers blocks rearrangement
Gene construct to test control elements: Deletion of any of Promoters (P or Pi) or Enhancers (Ei or 3’Eμ blocks recombination.
Vg6: TGG GAT A cactcta…………
Jg1: …………cactgtg AT AGC
P N P VJVJ1 TGG GAT T AGCVJ2 TGG GA AGCVJ3 TGG GA CCG AT AGCVJ4 TGG GAT TGG GCVJ5 TGG GA AT AT AGCVJ6 TGG GAT A T T AGCVJ7 TGG GAT TT AT AT AGC
Mechanism of action of RAG1 & RAG2
N addition by TdT=Terminal
5’ N-O-P-O-N 3’
3’ N-O-P-O-N 5’
several independent ways
Note: there are exons encoding the membrane and secreted forms of each of the antibody heavy chains.
Prior to activation, B cells express two forms of antibody as membrane receptors, IgM and IgD. On any given cell, the antigen specificity is identical.
This is accomplished by differential RNA splicing.
the immune response
Somatic hypermutation introduces diversity BCR (not in TCR)
Selection of clones with
the highest affinity
silent Phe ttc
only in B cells and not in T cells
1. Occurs at high rates: 10 -4 -10 -3 /bp/generation.
2. Occurs by untemplated single base substitutions.
3. Restricted to a brief period of B cell differentiation.
4. Restricted to the rearranged V region and its immediate
5. Occurs in germinal centers with T cell help.
6. Occurs throughout the V region but more frequently in
RGYW (A/G G C/T A/T) motifs.
BioEssays 20:227–234, 1998
Patricia J. Gearhart and Richard D. Wood
induces somatic hypermutation
removes base leaves sugar backbone
CSR occurs by an intrachromosomal deletional recombination between switch (S) region sequences located upstream of the constant region genes. S region sequences consist of tandem repeats of short (20–80 bp) consensus elements, extending from 2 to 10 kb in length, and recombination can occur at any site within the S regions. The process is thought to be initiated by the creation of double-strand breaks within the S regions, consistent with the ability to detect the deleted DNA as a circle. Although the different S regions have short sequence elements in common (e.g., GGGGT, GGGCT, or GAGCT), they differ too much to undergo homologous recombination and CSR is thought to occur by a type of nonhomologous end joining (NHEJ).
Activation of B lymphocytes by antigen and costimulatory signals, usually from T lymphocytes, initiates two processes of antibody diversification. Somatic hypermutation (SHM) introduces mutations in the variable region genes, which, in conjunction with antigen selection, generates antibodies with increased affinity.
The second process, class switch recombination (CSR), enables B Cells to diversify the constant region and thereby the effector function of the antibody molecule, while maintaining the same antigen-binding domain.
These two mechanisms have several shared features but both are poorly understood
Activation-induced cytidine deaminase (AID) is essential for both SHM and CSR
The data indicate its role is to convert dC to dU residues within variable genes and S regions. Resolution of the dU residues could introduce mutations that are characteristic both of SHM and of segments surrounding S–S junctions. Furthermore, removal of the dU residues by the base excision repair (BER) pathway could introduce the DNA breaks necessary to initiate CSR. This has not been demonstrated; however, CSR is 90% reduced in B cells from mice deficient in the BER enzyme uracil DNA glycosylase (UNG) that excises dU residues, and even more severely reduced in some patients with hyper IgM syndrome caused by deleterious mutations in UNG.
Cytokines direct transcription of I-S-CH which initiates CSR