Micro 297 Graduate Immunology Lecture 15 Development of B Lymphocytes I Generation of Antibody Diversity Thursday August 14, 2003 Michael Wolcott READING Chapter 7 - PowerPoint PPT Presentation

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Micro 297 Graduate Immunology Lecture 15 Development of B Lymphocytes I Generation of Antibody Diversity Thursday August 14, 2003 Michael Wolcott READING Chapter 7

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Title: Micro 297 Graduate Immunology Lecture 15 Development of B Lymphocytes I Generation of Antibody Diversity Thursday August 14, 2003 Michael Wolcott READING Chapter 7


1
Micro 297 Graduate ImmunologyLecture
15Development of B Lymphocytes IGeneration of
Antibody DiversityThursday August 14,
2003Michael WolcottREADING Chapter 7
2
Overview of B-Cell Development
3
Generation of Lymphocyte Antigen Receptor
  • The expression of the antigen receptor is the
    defining (and essential) event in the development
    of both B and T cells.
  • Antigen receptors, in the form of Igs on B cells
    and the TCR on T-cells, are the means by which
    lymphocytes sense the presence of antigen in
    their environment.
  • The diverse repertoire of lymphocyte receptors is
    accomplished through complex and elegant genetic
    mechanisms.
  • The basic mechanism for generation of diversity
    is common to both B cells and T cells and
    involves many if not all of the same enzymes.

4
Recall these Facts
  • The receptors produced by each lymphocyte have a
    unique antigen specificity which is determined by
    the structure of their antigen-binding site.
  • The wide range of antigen specificities in the
    antigen receptor repertoire is due to variation
    in the amino acid sequence in the V region.
  • Each individual possesses billions of
    lymphocytes, these cells collectively provide the
    individual with the ability to respond to a great
    variety of antigens.
  • In each chain the V region is linked to an
    invariant constant region which provides effector
    function

5
Genetic Model Compatible with Ig Structure
Any model must accommodate known properties of
Igs
  • The vast diversity of antibody specificities
  • The presence of a variable region and a constant
    region
  • The existence of different isotypes with the
    same antigen specificity
  • Germline Theory
  • Each antibody specificity coded by a germline
    (inherited) gene
  • What are the problems with this theory?
  • Somatic Mutation Model
  • Genome contains a small number of genes from
    which the diversity of antibody specificities is
    generated by mutation.
  • What are the problems with this theory?

6
Problems with the Germline and Somatic-Mutation
Models
  • Germline Theory
  • Part of gene subject to wide variation and part
    characterized by relative constancy.
  • So many specificities so few genes
  • Same variable regions on different isotypes
  • Somatic Mutation Theory
  • Part of gene subject to wide variation and part
    characterized by relative constancy.
  • Same variable regions on different isotypes
  • Resolution Begins
  • Dreyer-Bennent Hypothesis

7
The Dreyer Bennett Hypothesis
Two genes one polypeptide chain
  • Two separate genes encode a single Ig H or L
    chain
  • One gene for the V region and a separate gene for
    the C region
  • The two genes come together at the DNA level and
    are transcribed together
  • Thousands of V genes and a single C gene
  • Strength of this Recombination Model
  • Accommodates one part of the molecule varying
    with the other part remaining relatively constant
  • Accommodates how a single V region can be
    associated with more than one isotype
  • PROBLEM
  • So many specificities - so few genes!

8
Proof of Dryer-Bennett Hypothesis
Ig genes are rearranged in B cells
Conclusion
  1. Embryonic cells have the genes encoding the V
    region and C region considerably separated in the
    genome.
  2. During B cell development the genes are for V and
    C regions are brought much closer together.
  3. This simple experiment showed that segment of
    genomic DNA within the Ig genes are rearranged in
    cells of the B-lymphocyte lineage, but not in
    other cells.

9
Each V Region is Encoded by More Than One Gene
Segment
  • Cloning and sequencing of Ig genes showed even
    greater complexity that predicted by Dreyer and
    Bennett.
  • The DNA sequence encoding a complete V region is
    generated by the somatic (site specific)
    recombination of separate gene segments.
  • A single C gene segment encodes the C region

10
Gene Segments of VL and VH Regions
  • Light chain V region two gene segments
  • V gene segment first 95-101 amino acids
  • J (joining) gene segment up to 13 amino acids
  • Heavy chain V region three gene segments
  • V and J gene segments
  • D (diversity) gene segment

11
Organization of Ig-gene segments in the mouse
12
There are Multiple Different V-region Gene
Segments
  • The immunoglobulin gene segments are organized
    into three cluster or genetic loci the ?, ?,
    and heavy-chain loci each on a separate
    chormosome.
  • The V gene segments can be grouped into families
    in which each member shares at least 80 sequence
    identity with other in the family.
  • The families can be grouped into clans, made up
    of familes that are more similar to each other
    than to families in other clans.
  • VH gene segments identified from amphibians,
    reptiles, and mammals fall into three clans.
  • This suggests that these clans existed in a
    common ancestor of these modern animal groups.

13
V-region Genes are Constructed From Gene Segments
by Somatic (Site Specific) Recombination
  • VL by V-J recombination producing VJ variable
    region gene.
  • VH by D-J recombination followed by V to D-J
    recombination producing VDJ variable region gene.

14
Genetic RecombinationGeneral vs. Site Specific
  • Genetic recombination
  • the ability of DNA to undergo rearrangement that
    can vary the particular combinations of genes
    present in any individual genome.
  • General recombination
  • genetic exchange that takes place between any
    pair of homologous DNA sequences, usually located
    on two copies of the same chromosome e.g. the
    exchange of sections of homologous chromosomes in
    the course of meiosis.
  • Site specific recombination
  • DNA homology is not required. Instead, exchange
    occurs at short, specific nucleotide sequences
    that are recognized by site-specific enzymes
    examples integration of lambda phage in bacteria
    and somatic recombination of gene segments in
    Igs and TCRs.

15
5.4 Kappa Light Chain Gene Rearrangement
16
5.5 H-chain Gene Rearrangement
17
Rearrangement of V, (D), and J gene segments is
guided by flanking sequences
Recombination Signal Sequences RSS ensure
that DNA rearrangements take place at the correct
location relative to the V, D, or J gene segment
  • Conserved heptamer and nonamer sequences flank
    the gene segments
  • The spacer between the hepatmer and nonamer
    sequences is always approximately 12 bp or
    approximately 23 bp.
  • The spacer varies in sequence but its conserved
    length corresponds to one or two turns of the DNA
    double helix. This brings the heptamer and
    nonamer sequences to the same side of the DNA
    helix
  • The heptamer spacer nonamer is called a
    recombination signal sequence - RSS
  • 12/23 rule recombination, for the most part,
    only occurs between a 12 bp (one turn) and a 23
    bp (two turn) RSS

18
5.6 Recombination Signal Sequences
19
The reaction that combines V, D, and J gene
segments involves both lymphocyte-specific and
ubiquitous DNA-modifying enzymes
The complex of enzymes that act in concert to
effect somatic V(D)J recombination is termed the
V(D)J recombinase
  • The products of the two genes rag-1and rag-2
    (recombination-activating genes) comprise the
    lymphoid-specific components of the recombinase.
  • The remaining enzymes in the recombinase are
    ubiquitously expressed DNA-modifying proteins
    that are involved in DNA repair, DNA bending, or
    the modification of the ends of the broken DNA.
    These include DNA ligase, DNA-dependent protein
    kinase (DNA-PK), and Ku which is a heterodimer
    (Ku 70Ku 80) that associates with DNA-PK.

20
Enzymatic Steps in Recombination
21
V(D)J Recombination is a Multistep Process
  1. RAG protein complexes bind to 12 and 23 bp spaced
    RSS
  2. The protein complexes bind to each other bringing
    together the segments to be joined
  3. The DNA is cleaved to create a hairpin structure
    at the ends of the Ig gene segments
  4. Other DNA-modifying proteins bind to the hairpins
    and the cleaved RSS ends
  5. The DNA hairpins are cleaved at random.
    Additional bases may be added by TdT or
    subtracted by exonuclease to generate imprecise
    ends.
  6. DNA ligase IV joins the ends of the gene segments
    to for the coding joint and the RSS ends to form
    the signal joint.

22
5.7 Model for Recombination
23
Light Chain Recombination Can Occur By Either
looping out or Inversion
Which mechanism utilized depends on the
orientation of the V and J segments.
Opposite Orientation Inversion
Same Orientation looping out
24
Are RAG-1 And RAG-2 the only lymphoid specific
enzymes necessary for recombination of Ig or TCR
gene segments?
YES
So how would you prove it?
25
5.9 Experimental Identification or Rag1 and Rag2
26
Summary of the experimental identification of
RAG-1 and RAG-2
  • Retroviral construct containing a promoter
    sequence,V and J gene segments with flanking RSS
    and a gene that confers resistance to
    mycophenolic acid (in the opposite orientation of
    the promoter).
  • The orientation of the RSS sequences requires
    that rearrangement occurs by inversional
    recombination.
  • If the construct is rearranged then the
    resistance gene is brought into the same
    orientation as the promoter and the cells become
    resistant to mycophenolic acid.
  • A variety of cells were tested with this system
    and only pre- B cells and pre-T cells were able
    to rearrange the V and J segments.
  • However, fibroblasts could carry out the
    rearrangement if transfected with DNA coding
    RAG-1 and RAG-2.

27
5.10 Recombination Defects
28
5.11 Junctional Flexibility Productive vs.
Non-productive
29
Productive vs. Non-productiveRearrangements
  • The joining of the V(D)J gene segments is
    imprecise and gene segments can be joined out of
    phase, thus the triplet reading frame for
    translation is not preserved.
  • In such a non-productive rearrangement, the VJ or
    VDJ unit will contain numerous stop codons, which
    interrupt translation.
  • When joined in phase, the reading frame is
    preserved and thus is a productive rearrangement.

30
Generation of Antibody Diversity
  1. Multiple germline gene segments
  2. Combinatorial V-(D)-J joining
  3. Junctional flexibility
  4. P-region nucleotide addition (P-addition)
  5. N-region nucleotide addition (N-addition)
  6. Combinatorial association of light and heavy
    chains.
  7. Somatic hypermutation

31
5.14 Junctional Flexibility
32
5.15a P-Nucleotides
33
5.15b N-Nucleotides
34
5.16 Somatic Hypermutation
35
Summary The combination of many sources of
diversity generates a vast repertoire of antibody
specificities from a limited number of genes
  • Diversity with in the Ig repertoire is achieved
    by several means.
  • V regions are encoded by separate gene segments,
    which can be brought together by somatic
    recombination to make a complete V region gene.
  • Many V region gene segments are present in the
    genome, thus providing a heritable source of
    diversity.
  • Combinatorial diversity results from the random
    recombination of separate V , D and J gene
    segments to form a complete V region exon.
  • Variability at the joints is increased by
    N-region and P-region additions and by the
    variable deletion of nucleotides at the ends of
    coding sequences.
  • The association if different light and heavy
    chain V regions to form the antigen-binding site
    of an Ig molecule contributes further to the
    diversity.
  • Finally, after an immunoglobulin is expressed,
    the coding regions of the V regions are modified
    by somatic hypermutation following stimulation of
    the B cell by antigen.

36
Structural Variation in Immunoglobulin Constant
Regions
  • The immunoglobulin H-chain isotype are
    distinguished by the structure of their constant
    regions.
  • Antibody C-regions confer functional
    specialization.
  • Co-expression of IgM and IgD on B cells results
    from alternatively spliced H-chain transcripts.
  • Transmembrane and secreted forms of Igs are
    generated from alternative H-chain transcripts.
  • CLASS SWITCHING the same VH exon can associate
    with different CH genes in the course of an
    immune response.

37
Organization Of The Ig Heavy-chain C-region Genes
In Mice And Human
38
Co-expression of IgD and IgM is Regulated By RNA
Processing
39
Co-Expression of IgD and IgM
  • Mature B cells that co-express IgM and IgD on
    their surface have not undergone class switching.
    instead
  • In mature B cells, transcription initiated at the
    VH promoter extends through both Cµ and Cd exons.
  • The long primary transcript is then processed by
    cleavage and polyadenylation (AAA), and by
    splicing.
  • In this process there is no alteration at the DNA
    level.
  • The differential processing of the long mRNA
    transcripts is developmentally regulated.
    Immature B cells express only IgM mature B cells
    IgM and IgD activated B cells lose expression of
    IgD and express a single isotype of Ig.
  • The exact function of IgD on the surface of
    mature B cells is unclear. Gene-target mice
    lacking the delta exon appear to have normal
    immune responses.

40
Transmembrane and Secreted Forms of Igs
41
Transmembrane and Secreted Forms of Igs
Both forms are derived from the same H-chain gene
sequence
  • Each H-chain has
  • Two exons that encode the transmembrane region
    and the cytoplasmic tail
  • One exon that encodes the carboxy-terminus of the
    secreted form
  • The events that dictate whether a H-chain RNA
    will result in secreted or transmembrane occur
    during processing of the initial transcript.
  • The selection of transmembrane or secreted form
    is developmentally regulated. Prior to antigen
    stimualtion B cells make predominately the
    transmembrane form. However, plasma cell make
    exclusively the secreted form.

42
Isotype Switching Involves Recombination Between
Specific Switch Signals
43
Isotype Switching
  • Repetitive DNA sequences that guide isotype
    switching are found upstream of each of the
    C-region genes.
  • Switching occurs by recombination between these
    repetitive sequences (switch signals).
  • Isotype switching results in deletion of the
    intervening DNA
  • Since the intervening DNA is deleted back
    switches are not possible, but additional
    switches to down stream isotypes is possible
  • The initial switching event takes place from the
    µ switch region
  • Subsequent switches to other isotypes take place
    from the recombinant switch region formed after µ
    switching.
  • Isotype switching is unlike V(D)J recombination
    is several ways
  • All isotype switching is productive
  • It uses different recombination signal sequences
    and enzymes
  • It happens after antigen stimulation not during B
    cell development
  • The switching process is not random it is
    regulated by external signals from T cells

44
Isotype Switching Involves Recombination Between
Specific Switch Signals
45
Somatic Hypermutation
Somatic hypermutation further diversifies the Ab
repertoire
  • Introduces variation into the rearranged
    immunoglobulin V-region that is subject to
    positive and negative selection
  • Occurs in the germinal center following antigen
    stimulation of the B cell.
  • Somatic hypermutation requires signals from
    activated T cells
  • Hypermutation is thought to occur due to the
    introduction of double strand breaks in the DNA
    of V regions, followed by error prone repair.
  • Hypermutation occurs at a similar time to class
    switching, but appear to involve different
    enzymes and mechanisms.
  • We will cover hypermutation in more detail in the
    lectures on B cell activation.

46
Regulation of Ig-Gene Transcription
  • Immunoglobulin genes are expressed only in B
    cells
  • Genes are expressed at different rates during
    different stages of development
  • Three major classes of cis regulatory sequences
    in DNA regulate transcription of Ig-genes
  • Promoters short nucleotide sequences extending
    about 200 bp upstream from the start site, that
    promote intiation of RNA transcription
    orientation dependent.
  • Ehancers nucleotide sequences siturate some
    distance upstream or downstream from a gene that
    activate transcription from the promoter sequence
    in an orientation-independent manner
  • Silencers nucleotide sequences that
    down-regulate transcription, operating in both
    directions over a distance

47
Promoters, Enhancers and Silencers inH-Chain
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