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 (RSS’s)

•  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

Chapter 4 See Fig. 4-9 Page 88

Heavy chain

Light chain

Heavy chain

Light chain

See p97

Variable region, includes Heavy and light chains

One gene One protein

109 different antigens 109 antibody genes ???

The problem: Not enough DNA to go around!

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

See Figure 5-2, p114 “Tonegawa’s bombshell”

B cell Non-B cell

Southern Blot

The evidence for gene rearrangement

These gene fragments are far apart, separated by >>> DNA

p. 111

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

(Disregard λ, for now)

2. 1.

Delete large segments of DNA (irreversible!)

Similar process with light chain

VDJ recombination: (intervening DNA is removed and discarded)

DNA

pre-mRNA

mRNA

protein

Hint: Know this!

! Hint: Don’t know these exact numbers, but know how they were derived!

Number actually MUCH higher ! Junctional Diversity

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)

See Fig 5-6, page 119 Heptamer, spacer, nonamer Palendrome, …… AT rich

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

(Disregard Inv Join)

RAG = recombinase

Junctional Diversity

Junctional Flexibility

*

*

(many)

IgM IgD

IgM and IgD can be co-expressed ! (altern. RNA splicing!)

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

Class Switching - DNA

We Loop out 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).

FACSTM allows individual cells to be identified by their cell-surface antigens and to be sorted. General process is called flow cytometry

Antibodies conjugated with fluorochromes

Fluorescence intensity

Cel

l num

ber

Typical data from flow cytometry