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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

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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

Ch. 5


p. 112

Ch. 5


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

Ch. 5


p. 114

Ch. 5


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

Ch. 5


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

Ch. 5


p. 117

Ch. 5


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

Ch. 5


How does rearrangement occur?

Each V, D and J is flanked by RSS’s

(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

Ch. 5


p. 118

Ch. 5


p. 121

Ch. 5


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

Ch. 5


p. 122

Ch. 5


Generation of antibody diversity

(why are there so many possible antigen

combining sites?)

Many reasons…

Ch. 5


1. Multiple germline gene segments are inherited

In human germline:

51 VH, 27 D, 6 JH

40 V, 5 J 

30 V , 4 J

Ch. 5


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 (200+120) = 2.64 X 106 possible

combinations

(random combination of H and L chains)

Without taking into account other sources of

diversity

Ch. 5


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

Ch. 5


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

Ch. 5


p. 129

Ch. 5


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.

Ch. 5


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

Ch. 5


p. 131

Ch. 5



p. 134

Ch. 5



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

monoclonal antibodies

Ch. 5


Antibody genes and genetic engineering

Mouse mAb’s generate HAMA

(Human Anti-Mouse Ab)

Cleared quickly;

Allergic reactions

Now we have mouse CDR’s in human constant regions (“humanized Ab’s”)

Cattle and mice producing human Ab’s (due to HAC)

Bacteriophage libraries of Ab combining sites

Ch. 5


p. 137

Ch. 5


p. 138

Ch. 5




p. 139

Ch. 5


p. 139

Ch. 5





p. 141

Ch. 5


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)

Ch. 5


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

Ch. 5


Therapeutic applications

Cancer treatment

Imaging

Immunotoxins

Catalytic antibodies (Abzymes)?

Research applications

Structure-function analysis

Recombinant antibodies

“Humanized” antibodies

Ch. 5


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