Antibody generation- Chapter 3

(The B cell repertoire)

See p85, Chapter 3

The Big Picture

The remarkable range of antigens that our immune cells

can see is due to a random combination of gene segments
(VDJ) that occurs in B and T cells.
Recombination is performed by RAG1/2 complex,
recognizing specific DNA sequences (RSSs)
Additional diversity comes from imperfect joining of gene
segments
Alternative RNA splicing allows for co-expression of
different forms of antibody bya single B cell
Activated B cells can further modify the DNA of the
antibody genes: somatic hypermutation and class
switching

Heavy chain

Heavy chain
Light chain
Chapter 4
See Fig. 4-9
Page 88

Light chain

Variable region, includes

Heavy and light chains

See p97

The problem: Not enough DNA to go around!

One gene

One protein

109 different antigens

109 antibody genes ???

The solution: VDJ recombination

Generating Diversity: DNA

Recombination
In 1976, Hozumi and Tonegawa showed
that Ab-producing cells have different DNA
sequence than other somatic cells in Ig
locus - evidence for DNA recombination
(1987 Nobel Prize)
Sequencing of Ig genes has identified gene
segments that are brought together by
recombination

The evidence for gene rearrangement

B cell

Non-B cell

Southern Blot

See Figure 5-2, p114

Tonegawas bombshell

These gene fragments are far apart,

separated by >>> DNA

Today:
1. Gene (DNA) rearrangements
2. RNA splicing

p. 111

VDJ recombination: (intervening DNA is removed and discarded)

(Disregard , for now)

Similar process with light chain

2.

1.

Delete large segments of DNA (irreversible!)

DNA

pre-mRNA
mRNA

protein

Hint: Know this!

Number actually MUCH higher ! Junctional Diversity

! Hint: Dont know these exact numbers, but know how they were derived!

Ig Locus Rearrangement
Sequence-specific: recombination signal is a
conserved heptamer (CACAGTG), a spacer (12 or
23 non-conserved bases), and a conserved
nonamer (ACAAAAACC)
12/23 Rule: Recombination machinery always
joins a gene segment with a 12-bp spacer to
another with a 23-bp spacer
This ensures that the correct gene segments get
joined (no V-V, H chain always has D between V
and J, etc.)

The 12-23 Rule (one turn, two turn rule)

Heptamer, spacer, nonamer

Palendrome, AT rich

See Fig 5-6, page 119

V(D)J Recombination Machinery

Uses both specific (RAG1/2) and ubiquitous
factors
RAG1/2 complex is the sequence-specific
recombinase: recognizes recomb signal,
brings a 12 and a 23 signal together, and
cleaves DNA
Cleaved DNA is repaired by general DNA
repair factors

RAG = recombinase
(Disregard Inv Join)

Junctional Diversity

Junctional Flexibility

(many)

IgM and IgD can be

co-expressed !
(altern. RNA splicing!)
IgM

IgD

Surface Ig expression

RNA processing allows the co-expression of IgM and IgD

Refer to Figure 5-5, p118

RNA processing to form secreted and membrane IgG

Encodes
transmembrane
domain

Somatic Hypermutation
After rearrangement !!! - [ in response to antigen ]

Increased mutations
as response continues

Affinity maturation !

Immunoglobulin class switching

See Chapter 4

This process occurs at the level of DNA,

so it is irreversible

We Loop out DNA

Class Switching - DNA

Antibody conjugates to reveal the specific interaction between

antibodies and antigens
Attach antibodies to
1. Solid support-separate the solid from liquid.
Sepharose beads (immunoppt, affinity chromatography)
magnetic beads (cell separation)
plastic surface (ELISA)
2. Enzymes-reveal the interaction of antibodies and antigens
through enzymatic reactions (ELISA, ELIspot, Western blot,
immuno-histochemistry).
horseradish peroxidase
alkaline phosphatase
3. Fluorochromes-reveal the interaction of antibodies and
antigens through fluorescent light (FACS, IF microscopy).

Antibodies conjugated with fluorochromes

FACSTM allows individual cells to be identified by their cell-surface antigens and to be

sorted. General process is called flow cytometry

Cell number
Fluorescence intensity

Typical data from flow cytometry