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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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origin of diversity bibliography
Origin of diversityBibliography

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)

slide2

Clonal selection theory

יצירת מאגר של

לימפוציטים בשלים

תא אב יחיד מתמיין

למספר גדול של

לימפוציטים שלכל

אחד מהם ספציפיות

שונה

חלוקה והתמיינות

של לימפוציטים

ספציפיים ליצירת

קלון של תאים

אפקטוריים

סילוק של לימפוציטים

לא בשלים המגיבים

עם אנטיגנים עצמיים

Memory

cells

B-cells:

plasma cells

Ab secretion

T cells:

T-helper T-killer

figure 3 6
Figure 3-6

CDR1

CDR2

CDR3

CDR1

CDR2

CDR3

figure 4 2
Figure 4-2

The chance for successful rearrangement is 33%

ontogeny of the b lymphocyte
Ontogeny of the B Lymphocyte
  • Early differentiation
    • Many stages defined by Ig gene rearrangements
    • Progenitor cells
      • Hematopoietic stem cells
      • Pro-B
      • Pre-B
      • Immature B
pathway of b cell differentiation
Pathway of B cell Differentiation

Antigen Independent

Antigen Dependent

IgG “Memory” cell

Cytoplasmic

m chain

Heavy chain +

surrogate light

chain

“Heavy” plus

“light” chain

((חליפית)

Mature

IgM + IgD

Pre-Pro B

Pro B

Pre-B1

Pre-B2

B

“Plasmablast”

Immature

IgM

D--->JH

VH-->DJH

VL-->JL

ontogeny of the b lymphocyte1
Ontogeny of the B Lymphocyte
  • Process of differentiation
    • Signals that promote survival and proliferation of early cells
      • Adhesive interactions with stroma (non-lymphoid cells that make up marrow matrix)
      • Secretion of IL7
ontogeny of the b lymphocyte2
Ontogeny of the B Lymphocyte
  • Process of differentiation (continued)
    • Early cells
      • Pro-B – heavy chain D gene segment rearranges to J segment
      • Pre-B – Heavy chain V gene segment rearranges to join DJ region
        • Rearranged VDJ put close to heavy chain constant m gene
        • Synthesizes a m heavy chain
figure 7 4 part 1 of 3
Figure 7-4 part 1 of 3

V-DJ

rearrangement

D-J

rearrangement

ontogeny of the b lymphocyte3
Ontogeny of the B Lymphocyte
  • Pre-B cell – expresses mchain as a trans-membrane molecule at cell surface
    • In conjunction with products of two non-rearranged genes
      • Lambda 5 and VpreB
        • Function as surrogate light chains
    • Also in conjunction with additional trans-membrane molecules linked by disulfides
      • Ig alpha (CD79a)
      • Ig beta (CD79b)
    • Combination of tran-smembrane molecules (pre-B cell receptor; pre-BCR)
slide18

mheavy chain with surrogate light chains

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

Signal Transduction;

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

rearrangement

figure 7 6 part 2 of 2
Figure 7-6 part 2 of 2

Sensitive to self

antigens

ontogeny of the b lymphocyte4
Ontogeny of the B Lymphocyte
  • Immature B cells
    • L chains pair with mH chains to form monomeric IgM then inserted into membrane
    • Recognize Ag and respond to it
      • Long-lasting inactivation instead of expansion and differentiation
      • Interact with self-Ag in bone marrow – inactivate cell
        • Called negative selection
        • Important for development of self-tolerance in B-lineage
          • Cells with potential reactivity to self prevented from responding (central tolerance)
ontogeny of the b lymphocyte5
Ontogeny of the B Lymphocyte
  • Development of self-tolerance
    • Immature B cell exposed in bone marrow to:
      • Self-molecule on surface cells
        • Apoptosis (deletion)
      • Non-cell surface molecule (soluble Ag)
        • Cell is inactivated but not deleted (anergized)
      • Reactivation of VDJ recombinase (receptor editing)
        • Ig L chain genes undergo secondary rearrangement
          • Use unrearranged V or J segments
          • Generates specificity for non-self Ag – rescued from inactivation
slide27

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

slide28

Before clonal deletion of an anti-self B cell, the cell can attempt receptor editingof the light chain

Fig 7.26

RAGs expression

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”)

figure 4 15
Figure 4-15

Va are mixed with Vd

slide43

In a TCR a locus there are about 70 V gene segments and 60 J segments.

This provides for many attempts at a productive rearrangement. Rearrangements

stop when there is positive selection.

slide47

Mechanisms contributing to generation of primary antibody

diversity in humans

VxJx(D)

200

120

6000

Hxk

H-L chain associations

1,200,000

Hxl

720,000

levels of regulation of ig gene expression god
Levels of Regulation of Ig Gene Expression & GOD
  • V-(D)-J rearrangement
  • Class switch recombination (CSR)
  • Somatic hypermutation (SH)
  • Receptor editing\revision
v d j recombination rag
V(D)J Recombination : RAG

RAG 1 & 2 required

  • RAG 1 or RAG 2 K.O. mice
  • Also SCID in Man
    • No VDJ recombination (B or T-cells)
    • No dsDNA breaks
rag function
RAG function
  • RAG 1 & 2 transfected into fibroblasts + synthetic V-D-J substrate

Recombines exons

Transcription control

In synthetic substrate transfected into cell lines and mice, deletion of any of promoters or enhancers blocks rearrangement

transcription controls v d j recombination
Transcription Controls V(D)J Recombination

P

Pi

Pi

Lp

D

J

Ei

3’Eμ

v

Gene construct to test control elements: Deletion of any of Promoters (P or Pi) or Enhancers (Ei or 3’Eμ blocks recombination.

slide54

Nucleotide sequences of TCR V-gamma6 / J-gamma1 junction

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

slide55

Generation of diversity

Mechanism of action of RAG1 & RAG2

N addition by TdT=Terminal

deoxynucleotidyl transferase

slide58

RSS

RSS

5’ N-O-P-O-N 3’

3’ N-O-P-O-N 5’

slide61

Antibody protein sequence is altered in

several independent ways

  • Regulation of membrane vs secreted IgM RNA splicing
  • (Pre-immune) membrane IgM/IgD co-expression RNA splicing
  • AID dependent (activation induced cytidine deaminase):
  • Affinity maturation Somatic hyper-mutation
  • Isotype switch: Switch to IgG, IgA, IgE antibody H-chain C exons
  • Gene conversion
slide62

Note: there are exons encoding the membrane and secreted forms of each of the antibody heavy chains.

slide63

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.

IgM

IgD

slide65

Antibody protein sequence and bioactivity changes during

the immune response

High affinity

IgG

Low affinity

IgM

IgM

figure 4 9
Figure 4-9

Somatic hypermutation introduces diversity BCR (not in TCR)

Affinity maturation

Selection of clones with

the highest affinity

neutral

Lys aaa

Arg aga

Positive

Glu gaa

Gly gga

deleterious

Ser tca

Stop taa

silent Phe ttc

ttt

Mutations

slide67

Characteristics of Somatic Mutation

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

flanking sequences.

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

the mutation domain
The mutation Domain
  • Mutations largely confined to variable regions
  • Occur over a 1-2 kB region around the rearranged VJ gene segment
  • Found predominantly in CDRs but due to selection
slide69

Hyper-mutations spread in the V(D)J region

Patricia J. Gearhart and Richard D. Wood

figure 3 61
Figure 3-6

CDR1

CDR2

CDR3

CDR1

CDR2

CDR3

requirements for recruiting shm somatic hyper mutation
Requirements for recruiting SHM(Somatic Hyper Mutation)
  • In kappa locus both intronic and 3’ enhancer elements are required
  • Transcription is required, mutations decrease with distance from the promoter
  • Expression of AID (activation-induced cytidine deaminase) is essential
slide73

Activation Induced cytidine Deaminase (AID)

induces somatic hypermutation

A

T

C

G

AID

slide76

Base excision repairof G-T mismatch

removes base leaves sugar backbone

slide81

.

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).

class switch recombination
Class Switch Recombination
  • Transcription in switch region required
  • Cytokine directs transcription to I exon promoter
  • RNA/DNA hybrid of product flags target site for recombination
ig class switch recombination
Ig Class Switch Recombination

Evidence:

  • CSR blocked by deletion of :-
    • Splice site
    • I exon promoter
    • I exon
  • RNA/DNA hybrids identified
ig class switch recombination1
Ig Class Switch Recombination
  • RAG 1 & 2 NOT required
  • CSR requires:-
    • Activation-induced cytidine deaminase (AID, mice & hu.)
    • MSH2 (K.O. mice)
ig class switch recombination2
Ig Class Switch Recombination
  • AID initiates staggered dsDNA breaks
  • Cut by endonuclease
  • Breaks are repaired by error-prone DNA pol.
class switch recombination1
Class Switch Recombination
  • DNA breaks at switch sites are staggered
  • Breaks are repaired by error-prone DNA pol. & ligation
class switch recombination2
Class Switch Recombination

Evidence:-

  • AID- - hu. & AID or MSH2 K.O. mice  No CSR
slide93

Birds

Rabbits

Cows

Pigs

Sheep (SHM)

horses

somatic hypermutation shm class switch recombination csr
Somatic Hypermutation (SHM) & Class Switch Recombination (CSR)

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

slide98

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.

class switch recombination3
Class Switch Recombination

Cytokines direct transcription of I-S-CH which initiates CSR