Ch. 5. How can the immune system

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Ch. 5. How can the immune system recognize so
many different epitopes? Antibody H and L chains
are composed of gene segments Many unique
variable segments are inherited A limited
variety of constant region sequences are
used They must be rearranged into functional
genes before they can be transcribed
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Organization of Ig genes Germline DNA- gene
segments surrounded by noncoding regions These
are rearranged to form functional genes by a
cut and paste method Light chains- V domain
is composed of V and J segments C domain is
composed of C segment
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In Heavy chains- - V domain is composed of V,
D, and J segments - C domain is composed of
one C segment - Segments in V domains rearrange
first - A single V domain can join to one C,
then rearrange subsequently to join to
another C domain
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Multigene families Two types of L chains ? or
? In humans 40 V?, 5 J?, 1 C? Similar
number of ? genes in humans Heavy-chain gene
families are similar but more complex (also have
D segment) CH regions formed from exons
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Heavy chain DNA D-J and V-DJ rearrangements
occur separately On a mature B cell, both mIgM
and mIgD are expressed on the cell surface On
any one cell, mIgM and mIgD have same V
domains, but different C domains
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How does rearrangement occur? Each V, D and J is
flanked by RSSs (Recombination Signal
Sequences) Mechanism is controlled by RAG-1 and
RAG-2 (recombination-activating genes) proteins
and an enzyme, TdT (terminal deoxynucleotidyl
transferase) If any of these proteins is
defective, no mature B cells can form nor T
cells
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p. 121
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B cells are diploid and contain chromosomes from
both parents. However, heavy chain genes are
rearranged on only one chromosome, as are light
chain genes (on another chromosome). Therefore,
any one B cell will contain one VH and one VL (?
antigen specificity) How? Allelic exclusion
one allele gets turned off
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Generation of antibody diversity (why are there
so many possible antigen combining sites?) Many
reasons
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1. Multiple germline gene segments are inherited
In human germline 51 VH, 27 D, 6 JH 40
V?, 5 J ? 30 V ?, 4 J?
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2. Combinatorial V-J and V-D-J joining 57 V X
27 D X 6 J 8262 possible combinations for VDJ
joining (H chain) 40 V X 5J 200 possible V?
and 120 possible V? (L chain) 8262 X
(200120) 2.64 X 106 possible combinations
(random combination of H and L chains)
Without taking into account other sources of
diversity
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3. Junctional flexibility in V-J or V-D-J
junction 4. Additional nucleotides added at
junctions if a single-stranded region is created
during the joining process

5. Somatic hypermutation (AFTER Ag
stimulation) mutations occur AFTER
rearrangement tend to occur in CDR
regions affects antigen affinity (tends to
increase) called affinity maturation (late in
IR) occurs in B cells but not T cells
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Class switching After antigen stimulation,
heavy-chain DNA can rearrange so VDJ can join to
another isotype Cytokines help determine the
isotype IgG2a or IgG3 (mice) IFN-? IgM IL-2,
IL-4, IL-5 IgE IL-4
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Mature B cells express both mIgM and
mIgD Alternative RNA splicing to give IgM and
IgD - The VDJC?C? contains 4 polyadenylation
sites - mIgM or mIgD can be generated
depending on which polyadenylation site is used
Alternative RNA splicing to give
membrane-bound and secreted Igs Synthesis,
assembly, secretion of Igs then occurs.
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Regulatory elements of transcription Promoters
upstream of initiation site, promote init. of
RNA transcription in a specific
direction Enhancers activate transcription,
not in a specific direction Gene silencers
down-regulate transcription Gene rearrangement
brings enhancers close to the promoter they
influence
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p. 134
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What is a monoclonal antibody? Derived from a
single clone and specific for a single
epitope 1975- Kohler and Milstein developed
the hybridoma technique for developing monoclona
l antibodies

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Antibody genes and genetic engineering Mouse
mAbs generate HAMA (Human Anti-Mouse
Ab) Cleared quickly Allergic reactions Now we
have mouse CDRs in human constant regions
(humanized Abs) Cattle and mice producing
human Abs (due to HAC) Bacteriophage
libraries of Ab combining sites
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Behavior of monoclonal vs polyclonal
antibodies Monoclonal antibodies tend to have
high affinity Polyclonal antiserum will have
mixture of low and high affinity
antibodies Avidity vs. affinity Antibodies can
be cross-reactive (source of some autoimmune
disorders)
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Genetically-engineered monoclonal antibodies are
widely produced Advantages over hybridoma
technology can choose isotype as well as
specificity Can be expressed in a variety of
host cells non-lymphoid mammalian
cells bacteria (antibody fragments),
phage plants, yeast mice, cattle Mutations of
interest can be introduced
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Therapeutic applications Cancer
treatment Imaging Immunotoxins Catalytic
antibodies (Abzymes)? Research
applications Structure-function
analysis Recombinant antibodies Humanized
antibodies