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THE GENERATION OF DIVERSITY
The ability of the immune system to recognize antigens depends on the antibodies
generated by B cells, and on the antigen receptors expressed by T cells.
despite the differences of TCR and Abs the molecular and cellular processes which
generate diversity are very similar for each molecule
this topic will discuss the ways in which the great diversity of Abs and TCRs are
generated
we produce more types of antibody than there are genes in our genome
IMMUNOGLOBULIN VARIABILITY
Mechanisms involved in generation of antibody diversity:
1. Multiple Genes there is a large number of separate genes (V1Vn), each encoding one
V region domain
2. Somatic mutation a V gene mutates to produce different B cell clones
3. Somatic recombination a number of gene fragments (J1Jn) recombine to join the
main part of V region gene this results in a protein that contains elements encoded by
different gene segments
IMMUNOGLOBULIN GENE RECOMBINATION
The and light chains, as well as the heavy chains are encoded by separate multigene
families situated on different chromosomes.
in germ line DNA each of these multigene families contains coding sequences, called
gene segments, separated by noncoding regions
during B cell maturation, these genes are brought together to form functional
immunoglobulin genes.
the and chain families contain V, J, and C gene segments; the rearranged VJ
segments encode the variable region of the light chains.
the heavy chain families contain V, D, J and C gene segments; the rearranged VDJ
gene segments encode the variable region of the heavy chain
-the C gene segments encode the constant regions
each V gene segment is preceded by a short signal or leader peptide that guides the
heavy or light chain through the endoplasmic reticulum; the signal peptide is cleaved
from the nascent heavy and light chain before assembly of the finished Ig molecule
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Light chain genes recombine V and J segments to make a gene for the V domain
Two separate gene segments were found to code for the V and C regions and in the
germ line (cells that are not producing Ab), these two gene segments were found to be
far apart on the chromosomes.
in contrast, in Abproducing cells the gene segments were brought closer together
The V and C fragments do not directly join but are separated by a joining gene segment,
the J segment
Structure of the
chain system The V gene segment codes for the V region of the Ab
light chain, up to amino acid 95; the J gene segment codes for the rest of the variable
region
in the mouse, there are 3 V segments, 4 J gene segments and 4 C gene segments
Structure of the
chain system this multigene family contains approximately 300 V
gene segments, 5 J gene segments and 1 C gene segment (Fig.)
Heavy chain genes recombine V, D and J segments to make a gene for the VH
domain
The heavy chain region is also encoded by V and J segments; additional diversity is
provided by a third gene segment, the D (diversity) gene segment (Fig)
the D gene segments encode for amino acids within the complementarity determining
region (CDR3)
in mouse, there are over 1000 VH gene segments, 30 D gene segments, 6 functional JH
gene segments followed by a series of CH gene segments
each CH gene segment encodes the constant region of the Igheavy chain isotype
Recombination sequences flanking the V, D and J gene segments direct joining of
the gene segments
recombination of gene segments is a key feature of the generation of a functional gene
for both the light and heavy chain variable regions
Unique recombinational signal sequences (RSSs) flank each germ line V, D, and J
gene segment; signal for recombination process
one RSS is located 3' to each V gene segment, 5' of each J gene segment and on both
sides of each D gene segment.
each RSS contains a conserved palindromic heptamer and a conserved ATrich nonamer
sequence separated by an intervening sequence of 12 or 23 base pairs (Fig,)
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the 12 and 23 base pair sequence correspond respectively, to one and two turns of the
DNA helix; for this reason they are referred to as oneturn signal sequences and two
turn signal sequences
signal sequences having a oneturn spacer can only join with sequences having a two
turn spacer (this is the oneturn/twoturn joining rule)
a two turn spacer occurs in the signal sequence of the VH and JH gene segments, and a
one turn spacer occurs in the signals on either side of the DH gene segment; this joining
rule ensures that the VH, JH, and DH gene segments join in a proper order and that
segments of the same type do not join with each other
VDJ recombination which takes place at the junctions between the RSSs and coding
sequences is catalyzed by enzymes collectively referred to as V(D)J recombinase
two enzymes which are required in VDJ recombination are RAG1 and RAG2
proteins and the enzyme terminal deoxynucleotidyl transferase (TdT)
mice that are RAG1 and RAG2 deficient have a defect in VDJ recombination and
cannot generate functional B or T cells; these mice are called SCID mice (severe
combined immunodeficiency)
The place at which V and J segment genes join may vary slightly
slight variations in the positions at which recombination takes place generates additional
diversity.
ie. the 95 th AA is encoded by the last codon of the V segment;
the 96 th AA is encoded by the first codon of the J segment (Fig.)
Sometimes, the 96 th AA is encoded in part by bases of the intron that is 3' to the V
segment and in part by bases of the first codon of the J segment
this will lead to variation in AA sequence
in addition, a few nucleotides may be inserted between the VH and D and between the
D and JH segments by means of the enzyme TdT (Fig)
because addition occurs without a need for a coding template these nucleotides are
called Nnucleotides (for nontemplate) and it is called Nregion diversity
SOMATIC MUTATION
Once the variable region has been rearranged, additional diversity can be generated by
somatic hypermutation
individual nucleotides are replaced with alternative nucleotides thus altering the
specificity of the encoded Ig; this only occurs after exposure to antigen
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somatic mutation is targeted to the V region segments and occurs at a frequency of 103
per base pair per generation (1 in every 1000 base pairs is mutated, this is much higher
than the spontaneous mutation rate of 1 in 109 bases) thus it is called hypermutation
somatic mutation is random, and will generate Abs with varying affinity for antigen
following exposure to antigen, B cells with higher affinity receptors will be
preferentially selected because of their greater ability to bind antigen
Additional diversity is generated as virtually any light chain may pair with any
heavy chain
To summarize:
Five mechanisms for the generation of antibody diversity:
1. multiple germ line V genes
2. VJ and VDJ recombination
3. recombinational inaccuracies
4. somatic point mutations
5. assorted heavy and light chains
CLASS SWITCH
- initially mature B cells express both IgM and IgD; How? a single mRNA consisting of
VDJ CμCδ is initially transcribed; then through alternate splicing results in
separate VDJ Cμ and VDJ Cδ mRNAs
following stimulation of a B cell, the heavychain DNA can undergo a further
rearrangement in which the V D J unit of the heavy chain can combine with any CH
gene segment
the exact mechanism of this process called class switching is unclear
it appears that there are DNA flanking sequences (switch sites, S) located upstream of
each CH segment (except C) that may be involved
-“Switch recombinase” is the enzyme that catalyzes the rearrangement of the VDJ
genes during class switching; Gene recombination is a proposed mechanism
recombination occurs between the switch sites, bringing the C region close to the VDJ
2. interaction of the CD40 protein on the B cell with CD40 ligand protein on the
helper T cell. For example, in hyper-IgM syndrome, the failure to interact properly
results in an inability of the B cell to switch to the production of IgG, IgA, or IgE.
Therefore, only IgM is made.
Note that once a B cell has “class” switched past a certain H chain gene, it can no
longer make that class of H chain because the intervening DNA is excised and
discarded. Class switching occurs only with heavy chains; light chains do not undergo
class switching
Allelic exclusion
A single B cell expresses only one L chain gene (either κ or λ) and one H chain gene. In
theory, a B cell could express two sets of immunoglobulin genes, a maternal set and a
paternal set. But this is not what happens. Only one set of genes is expressed, either
maternal or paternal, and the other set is silent (i.e., it is excluded). This is
called allelic exclusion. Successful rearrangement of the genetic material from one
chromosome results in the shutting down of rearrangement of genetic material from the
second chromosome
- If no successful rearrangement occurs, rearrangement of genetic material on the second
chromosome takes place. If no successful rearrangement occurs on either chromosome,
the cell dies.
Each individual contains a mixture of B cells, some expressing the paternal genes and
others the maternal ones. The mechanism of this exclusion is unknown although
methylation is thought to be the mechanism of silencing.
. Membrane and secreted Igs are produced by differential splicing of RNA
transcripts of heavy genes
the antigen receptor of the membrane bound and secreted Ig is identical except for an
extra stretch of AAs at the C terminus of each heavy chain
differential splicing gives rise to each form of Ig
STAGES OF IMMUNOGLOBULIN PRODUCTION
1. heavy chain rearrangement of D to J
2. addition of V gene segment
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3. transcription of VDJ
4. translation produces membrane and chains
5. the chain induces recombination of the light chain VJ
6. transcription of VJ or VJ
7. translation of and
8. both membrane IgM and IgD are present on the B cell
9. after primary antigen stimulation, the cell produces secreted IgM
10. after stimulation with a Tdependent antigen and with T cell help (cytokines), the cell
can switch classes to produce either IgG, IgA, or IgE which is accompanied by somatic
mutation of the V domains
GENES OF THE TCR
The TCR is generated by 4 sets of genes; the and expressed mainly in peripheral T
cells, and the and expressed in a minor population of peripheral T cells
arrangement of TCR genes is remarkably similar to the Ig heavy chains having V, D, J,
and C gene segments