Download Overview of B-Cell Development

Micro 297 Graduate Immunology
Lecture 15
Development of B Lymphocytes I
Generation of Antibody Diversity
Thursday August 14, 2003
Michael Wolcott
READING
Chapter 7
Overview of B-Cell Development
Bone Marrow
Periphery
Y
IgM
Y
IgM
IgD
Y
Y
Antigen
Y Y
Y Y
IgM
Plasma Cell
Lymphoid
Stem Cell
ProB Cell
PreB Cell
Immature
B Cell
Mature
B Cell
Y
Activated
B Cell
Memory
B Cell
Antigen Independent
Primary Lymphoid Organ
Antigen Dependent
Secondary Lymphoid Organs
Y
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 Ig’s 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.
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
Genetic Model Compatible with Ig Structure
Any model must accommodate known properties of Ig’s
1.
2.
3.
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?
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
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!
Proof of Dryer-Bennett Hypothesis
Ig genes are rearranged in B cells
Conclusion
Southern Blot
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 Blymphocyte lineage, but not in other
cells.
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
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
Organization of Ig-gene segments in the mouse
There are Multiple Different V-region
Gene Segments
• The immunoglobulin gene segments are organized into three
cluster or genetic loci – the κ, λ, 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.
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.
Genetic Recombination
General 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 Ig’s and TCR’s.
5.4 Kappa Light Chain Gene Rearrangement
5.5 H-chain Gene Rearrangement
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
5.6 Recombination Signal Sequences
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 70:Ku
80) that associates with DNA-PK.
Enzymatic Steps in Recombination
V(D)J Recombination is a Multistep Process
1.
2.
3.
4.
5.
6.
RAG protein complexes bind to 12 and 23 bp spaced RSS
The protein complexes bind to each other bringing together
the segments to be joined
The DNA is cleaved to create a hairpin structure at the ends
of the Ig gene segments
Other DNA-modifying proteins bind to the hairpins and the
cleaved RSS ends
The DNA hairpins are cleaved at random. Additional bases
may be added by TdT or subtracted by exonuclease to
generate imprecise ends.
DNA ligase IV joins the ends of the gene segments to for the
coding joint and the RSS ends to form the signal joint.
5.7 Model for Recombination
Light Chain Recombination Can Occur By
Either “looping out” or “Inversion”
Which mechanism utilized depends on the orientation of the V and J segments.
Same Orientation
Opposite Orientation
“looping out”
“Inversion”
Intervening
DNA “looped
out” and lost at
the next cell
division
Recombination of gene
segments that are in
the opposite orientation results in inversion and integration of
the intervening DNA
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?
5.9 Experimental Identification or Rag1 and Rag2
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.
5.10 Recombination Defects
5.11 Junctional Flexibility:
Productive vs. Non-productive
Productive vs. Non-productive
Rearrangements
• 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.
Generation of Antibody Diversity
1.
2.
3.
4.
5.
6.
Multiple germline gene segments
Combinatorial V-(D)-J joining
Junctional flexibility
P-region nucleotide addition (P-addition)
N-region nucleotide addition (N-addition)
Combinatorial association of light and heavy
chains.
7. Somatic hypermutation
5.14 Junctional Flexibility
5.15a P-Nucleotides
5.15b N-Nucleotides
5.16 Somatic Hypermutation
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.
1.
V regions are encoded by separate gene segments, which
can be brought together by somatic recombination to make a
complete V region gene.
2.
Many V region gene segments are present in the genome,
thus providing a heritable source of diversity.
3.
Combinatorial diversity results from the random
recombination of separate V , D and J gene segments to form
a complete V region exon.
4.
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.
5.
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.
6.
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.
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 Ig’s 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.
Organization Of The Ig Heavy-chain C-region Genes In
Mice And Human
Co-expression of IgD and IgM is Regulated By RNA
Processing
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 Cδ 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.
Transmembrane and Secreted Forms of Ig’s
Transmembrane and Secreted Forms of Ig’s
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.
Isotype Switching Involves Recombination Between
Specific Switch Signals
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
Isotype Switching Involves Recombination Between
Specific Switch Signals
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.
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 orientationindependent manner
– Silencers – nucleotide sequences that down-regulate
transcription, operating in both directions over a distance
Promoters, Enhancers and Silencers in
H-Chain