Antibody Diversity

Problem…the immune system

makes over one billion different
antibody proteins
• In 1950’s: central dogma stated DNA—to RNA—to
protein
• One gene for each protein
• Required millions of genes just for the immune
system
• Does not seem possible, but most scientists thought
it might be
• Today we know the human genome is less than
30,000 genes
• So, what is really going on???
Current theory must account for the
following known properties of
antibodies
B lymphocyte development
B lymphocyte development (2)
 Germ-line vs somatic-variation
theories
• Germ-line: stated that each antibody had its
own gene….nothing special, but required billions
of genes to account for numbers of antibodies
• Somatic-variation: some mutation and
recombination created vast number of genes for
antibody formation
• This introduced a new concept: targeted
mutation or recombination of DNA: is it
possible??
• Paradox: how could stability be maintained in C
region and diversity exist in V region?
 Dreyer and Bennett
• In 1965 proposed radical theory to account for
diversity of antibodies
• Each antibody was coded for by two separate
genes
• One for the variable region
• One for the constant region
• Combined at the DNA level and expressed
single mRNA
• Suggested 1000’s of variable region genes and
only one constant region gene
• Close, but no cigar
• Most biomedical scientists did not like this idea
and rejected it
 Tonegawa’s demonstration
• 1976—used restriction enzymes and DNA probes to show that
germ cell DNA contained several smaller DNA segments
compared to DNA taken from developed lymphocytes
(myeloma cells)
Genes for immunoglobulin proteins are
found on different chromosomes
Multigene organization of Ig genes
Kappa light chain rearrangement
Heavy chain rearrangement
 Mechanism of variable region
rearrangements
• Each V, D and J segments of DNA are flanked
by special sequences (RSS—recombination
signal sequences) of two sizes
• Single turn and double turn sequences (each
turn of DNA is 10 base pairs long)
• Only single turn can combine with a double turn
sequence
• Joining rule ensures that V segment joins only
with a J segment in the proper order
• Recombinases join segments together
 P and N region nucleotide alteration
adds to diversity of V region
• During recombination some nucleotide bases are cut
from or add to the coding regions (p nucleotides)
• Up to 15 or so randomly inserted nucleotide bases are
added at the cut sites of the V, D and J regions (n
nucleotides_
• TdT (terminal deoxynucleotidyl transferase) a unique
enzyme found only in lymphocytes
• Since these bases are random, the amino acid
sequence generated by these bases will also be
random
 N nucleotide addition at joining
segments: the addition of random
bases
Randomness in joining process helps
generate diversity by creating
hypervariable of antigen binding site
Imprecise joining generates
diversity
Some rearrangements are productive, others
are non-productive: frame shift alterations are
non-productive
Allelic exclusion: only one chromosome
is active in any one lymphocyte
7 means of generating antibody
diversity
Diversity calculations
 Somatic hypermutation adds even
more variability
• B cell multiplication introduces additional
opportunities for alterations to rearranged
VJ or VDJ segments
• These regions are extremely susceptible
to mutation compared to “regular” DNA,
about one base in 600 is altered per two
generations of dividing (expanding)
lymphocyte population
 Combination of heavy and light
chains adds final diversity of
variable region
• 8262 possible heavy chain combinations
• 320 light chain combinations
• Over 2 million combinations
• P and N nucleotide additions and
subtractions multiply this by 104
• Possible combinations over 1010
Location of variability occurs within
CDR regions of V domains (antigen
binding sites)
Class switching among constant regions:
generation of IgG, IgA and IgE with same
antigenic determinants—idiotypes
 Synthesis,
assembly and
secretion of
immunoglobulins
 Regulation of Ig gene transcription
Each lymphocyte rearranged gene has regulatory sequences that
control gene expression
Promoters: initiation sites of RNA transcription
Enhancers: upstream of downstream that transcription from the
promoter sequence
Silencers: down-regulate transcription in germline cells
Gene rearrangement brings enhancer and promoter regions close
together and eliminates silencer regions allowing transcription
 Understanding of immunoglobulin
structure and formation has opened
up a new world of possibilities
• Monoclonal antibodies
• Engineering mice with human immune
systems
• Generating chimeric and hybrid antibodies
for clinical use
• Abzymes: antibodies with enzyme
capability