Corso di Immunologia A.A. 2008-09 Genetica …

1

Corso di Immunologia

A.A. 2008-09

Genetica immunoglobuline

Generation of diversity (GOD-I)

2

The organization and expression of the immunoglobulin gene families.

The origins of antibody diversity.

KEYWORDS:

V gene, C gene, J region, D region, Leader, Enhancer, Promoter, Antibody

diversity, Germ line theory, Somatic mutation theory, N region insertions,

Junctional diversity, Combinatorial association, Multispecificity, Clonal

selection

Cosa dovremmo conoscere alla fine di questa lezione

3

A. Schematic diagram of a secreted IgG molecule. The antigen-binding sites are formed by the juxtaposition of VL and VH domains. The heavy chain C regions end in

tail pieces. The locations of complement- and Fc receptor-binding sites within the heavy chain constant regions are approximations. B. Schematic diagram of a

membrane-bound IgM molecule on the surface of a B lymphocyte. The IgM molecule has one more CH domain than IgG, and the membrane form of the antibody has C-

terminal transmembrane and cytoplasmic portions that anchor the molecule in the plasma membrane. C. Structure of a human IgG molecule as revealed by x-ray

crystallography. In this ribbon diagram of a secreted IgG molecule, the heavy chains are colored blue and red, and the light chains are colored green; carbohydrates are

shown in gray. (Courtesy of Dr. Alex McPherson, University of California, Irvine.)

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© 2005 Elsevier

Structure of an antibody molecule

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Figure 4-24 Terminologia necessaria

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The illustration depicts the changes in the structure of antibodies that may be produced by the progeny of activated B cells (one clone) and the related changes in

function. During affinity maturation, mutations in the V region (indicated by red dots) lead to changes in fine specificity without changes in C region-dependent effector

functions. Activated B cells may shift production from largely membrane-bound antibodies containing transmembrane and cytoplasmic regions to secreted antibodies.

Secreted antibodies may or may not show V gene mutations (i.e., secretion of antibodies occurs before and after affinity maturation). In isotype switching, the C regions

change (indicated by color change from blue to orange) without changes in the antigen-binding V region. Isotype switching is seen in membrane-bound and secreted

antibodies. The molecular basis for these changes is discussed in Chapter 10.

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© 2005 Elsevier

Changes in antibody structure during humoral immune responses.

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Background Due geni per una proteina.

Nel 1965 Dreyer e Bennet ipotizzarono che le regioni V e C delle

immunoglobuline fossero codificate da 2 geni distinti e tale

ipotesi é stata confermata sperimentalmente da Tonegawa.

Lo studio della variabilità dei geni delle immunoglobuline

rappresenta un bellissimo modello di ricombinazione

somatica.

7

History

Amino acid sequencing data revealed that a single C region

could be associated with many different V regions. Also, it was

shown that a single idiotype could be associated with different

C regions (eg. IgM and IgG). To explain these data it was

suggested that perhaps the two regions of the Ig molecule

were coded by separate genes and that the V and C region

genes were somehow joined before an Ig molecule was made

(i.e. there were two genes for one polypeptide).

8

History

This was a revolutionary concept but with the advent of

recombinant DNA technology, it has been shown to be the

correct. The Ig heavy and light chains are coded by three

separate gene families each one on a separate chromosome -

one for the heavy chain and one for each of the light chain

types. Each of these gene families has several V region genes

and one or more C region genes. The V and C regions genes

are not however immediately adjacent to each other.

9

Immunoglobulin Genetics History

• Same C region could associate with many V regions

– IgG Ab with different specificities

• Same V region could associate with many C regions

– Same idiotype on IgG and IgM Ab

• V and C regions coded for by separate genes

10

11

12

13

14

15

16

Immunoglobulin Genetics Germ line gene organization

• The organization of the kappa and lambda light

chain genes in the germ line or undifferentiated

cells is depicted in the next Figure.

17


Lambda light chains

• The lambda gene family is composed of 4 C

region genes, one for each subtype of 8 chain,

and approximately 30 V region genes. Each of the

V region genes is composed of two exons, one (L)

that codes for a leader region and the other (V)

that codes for most of the variable region.

Upstream of each of the C genes there is and

additional exon called J (joining). The L, V, J and

C exons are separated by introns (intervening

non-coding sequences).

18


Kappa light chains

• The kappa light chain gene family contains only

one C region gene, since there is only one type of

kappa light chain. There are many V region genes

(approximately 250) each of which has a leader

exon and a V exon. In the 6 gene family there are

several J exons located between the V and C

genes. All of the exons are separated by introns.

19

Light Chain Gene Families Germ line gene organization

J

1

C

1

E

J

2

C

2

E

J

3

C

3

E

J

4

C

4

E P

L V

1

P

L V

n

P

L V

2

Lambda light chain genes; n=30

P

L V 1

P

L V n

P

L V 2 J

2 J

3 J

5 C

E

J1 J

4

Kappa light chain genes; n=300

20

• As a cell differentiates into a mature B cell that will

make a light chain, there is a rearrangement of

the various genes (exons) and the gene begins to

be expressed. As a cell commits to become a B

cell making a light chain, there is a rearrangement

of the genes at the DNA level such that one of the

V genes is brought next to one of the J regions.

Light Chain Gene Families Gene rearrangement and expression

21

• This occurs by a recombination event which

removes the intron between the V and J regions.

The selection of which V gene is used is not

totally random; there is some preference for the

use of V genes nearest to the J regions. However,

with time all V genes can be used so that all

combinations of V genes and J regions can be

generated.


22


L V 1 L V

n L V

2 J

2 J

3 J

5 C

J

1 J

4

DNA P P P E

P P E

L V 1 L J

5 C

V 2 J

4

DNA

DNA Rearrangement

V C J

V C

Protein

Transport to ER

Protein

Translation C L V J

E

L V C

J

RNA

Transcription

Primary transcript

C L V J RNA Processing

RNA mRNA

23

• A consequence of this DNA rearrangement is that

the gene becomes transcriptionally active

because a promoter (P), which is associated with

the V gene, is brought close to an enhancer (E),

which is located in the intron between the J and C

regions.


24

• As transcription initiates from the promoter a pre-

mRNA is made which contains sequences from

the L, V J and C regions as well as sequences for

the introns between L and V and between J and

C. This pre-mRNA is processed (spliced) in the

nucleus and the remaining introns are removed.

The resulting mRNA has the L, V J and C exons

contiguous.


25


L V 1 L V

n L V

2 J

2 J

3 J

5 C

J

1 J

4

DNA P P P E

P P E

L V 1 L J

5 C

V 2 J

4

DNA

DNA Rearrangement

V C J

V C

Protein

Transport to ER

Protein

Translation C L V J

E

L V C

J

RNA

Transcription

Primary transcript


RNA mRNA

26

• The mRNA is translated in the cytoplasm and the

leader is removed as the protein is transported

into the lumen of the endoplasmic reticulum. The

light chain is assembled with a heavy chain in the

endoplasmic reticulum and the Ig is secreted via

the normal route of secretory proteins. The region

V region of the mature light chain is coded for by

sequences in the V gene and J region and the C

region by sequences in the C gene.


27


L V 1 L V

n L V

2 J

2 J

3 J

5 C

J

1 J

4

DNA P P P E

P P E

L V 1 L J

5 C

V 2 J

4

DNA

DNA Rearrangement

V C J

V C

Protein

Transport to ER

Protein

Translation C L V J

E

L V C

J

RNA

Transcription

Primary transcript


RNA mRNA

28

Heavy Chain Gene Family Germ line gene organization

Introns separate exons coding for H chain domains

Heavy chain genes; Vn=1000, Dn=15

P

L V1

P

L Vn

P

L V2 D2 D3 D1 Dn

C

C

C

3

C

C

2

C

1

C

4

C

1

C

2

J2 J3 J5 J1 J4

E

CH1 H CH2 CH3 CH4

29

• In the heavy chain gene family there are many C

genes, one for each class and subclass of Ig.

Each of the C genes is actually composed of

several exons, one for each domain and another

for the hinge region. In the heavy chain gene

family there are many V region genes, each

composed of a leader and V exon.


30

• In addition to several J exons, the heavy chain

gene family also contains several additional exons

called the D (diversity) exons. All of the exons are

separated by introns.


31


Introns separate exons coding for H chain domains

Heavy chain genes; Vn=1000, Dn=15

P

L V1

P

L Vn

P

L V2 D2 D3 D1 Dn

C

C

C

3

C

C

2

C

1

C

4

C

1

C

2

J2 J3 J5 J1 J4

E

CH1 H CH2 CH3 CH4

32

Heavy Chain Gene Family Gene rearrangement and expression

P

L V1

P

L Vn

P

L V2 D2 D3 D1 Dn C

C

J2 J3 J5 J1 J4

E

DJ rearrangement

P

L Vn

P

L V1

P

L V2 D2

D1 C

C

J5 J4

E DNA

VDJ rearrangement

D2 C

C

J5 J4

E P

L V1

P

L V2

DNA

Transcription

D2 C

C

J5 J4

E

L V2

RNA Primary transcript

33

• As a cell differentiates into a mature B cell that will

make a heavy chain, there is a rearrangement of

the various genes segments (exons) and the gene

begins to be expressed.


34

• As a cell commits to become a B cell making a heavy

chain, there are two rearrangements at the DNA level.

First, one of the D regions is brought next to one of the J

regions and then one of the V genes is brought next to

the rearranged DJ region. This occurs by two

recombination events which remove the introns between

the V, D and J regions. As with the light chains the

selection of the heavy chain V gene is not totally random

but eventually all of the V genes can be used.


35


P

L V1

P

L Vn

P

L V2 D2 D3 D1 Dn C

C

J2 J3 J5 J1 J4

E

DJ rearrangement

P

L Vn

P

L V1

P

L V2 D2

D1 C

C

J5 J4

E DNA

VDJ rearrangement

D2 C

C

J5 J4

E P

L V1

P

L V2

DNA

Transcription

D2 C

C

J5 J4

E

L V2


36

• A consequence of these DNA rearrangements is that the

gene becomes transcriptionally active because a

promoter (P), which is associated with the V gene, is

brought close to an enhancer (E), which is located in the

intron between the J and C: regions. As transcription

initiates from the promoter a pre-mRNA is made which

contains sequences from the L, V, D, J C: and C*

regions as well as sequences for the introns between L

and V, between J and C:, and between C: and C*.


37


P

L V1

P

L Vn

P

L V2 D2 D3 D1 Dn C

C

J2 J3 J5 J1 J4

E

DJ rearrangement

P

L Vn

P

L V1

P

L V2 D2

D1 C

C

J5 J4

E DNA

VDJ rearrangement

D2 C

C

J5 J4

E P

L V1

P

L V2

DNA

Transcription

D2 C

C

J5 J4

E

L V2


38

• The pre-mRNA is processed (spliced) in the nucleus and

the remaining introns, including those between the

exons in the C genes, are removed. The pre-mRNA can

be processed in two ways, one to bring the VDJ next to

the C gene and the other to bring the VDJ next to the C*

gene. The resulting mRNAs have the L, V, D, J and C: or

C* exons contiguous and will code for a mu and a

gamma chain, respectively.


39


E Primary transcript

E

J4 D2 V2 C

C

J5 L C

C

J5 L D2 J4 V2

RNA Processing

An

mRNA for C

C

L D J V C

L D J V

An

mRNA for C

Translation

C

heavy chain

C

L D J V C

L D J V

C

heavy

chain

Transport to ER

C

heavy chain

V C

C

D J V C

D J V

C

heavy

chain

V C

40

• The mRNAs are translated in the cytoplasm and the

leader is removed as the protein is transported into the

lumen of the endoplasmic reticulum. The heavy chain is

assembled with a light chain in the endoplasmic

reticulum and the Ig is secreted via the normal route of

secretory proteins. The region V region of the mature

heavy chain is coded for by sequences in the V gene, D

region and J region and the C region by sequences in

the C gene.


41


E Primary transcript

E

J4 D2 V2 C

C

J5 L C

C

J5 L D2 J4 V2

RNA Processing

An

mRNA for C

C

L D J V C

L D J V

An

mRNA for C

Translation

C

heavy chain

C

L D J V C

L D J V

C

heavy

chain

Transport to ER

C

heavy chain

V C

C

D J V C

D J V

C

heavy

chain

V C

42

43

Generation of diversity (1)

• I geni per le catene H, kappa e lambda (L) sono su cromosomi diversi (12,

16, 6 nel topo e 14, 2, 22 nell'uomo) e un primo livello di variabilita`

consiste nella scelta di unire una particolare catena H con una L (livello a).

• I geni V sono composti di 2 subunita` per L (chiamate V e J) e di 3 per H

(chiamate V, D, J, situate in quest'ordine lungo il cromosoma). La subunita`

J e` posta a valle di V ed a monte di C: la sua funzione e` di congiungere

(joining) V a C. La subunita` D prende il nome da "Diversity". Recenti

approssimazioni suggeriscono che vi sono circa 300 geni VH, 300 Vk, 2 Vl

(questo spiega in parte perche` la maggior parte delle Ig sono composte

dalla catena L kappa e non L lambda), 20 geni DH, 5 geni JH, Jk e Jl. Quindi

tramite ricombinazione casuale possiamo ottenere milioni di catene (livello

b) con diverse regioni V.

44

Generation of diversity (2) Nelle cellule non differenziate i geni che codificano per V, J e C (catene L) e i

geni che codificano per V, J, D e C (catene H) sono distanti ed il

differenziamento comporta una delezione di gran parte del DNA frapposto

con finale riarrangiamento dei geni V, J, D e C che verranno a trovarsi

vicino ma non attigui per la presenza di introni. Tale riarrangiamento rende

il gene trascrizionale attivo. Il complesso V-J-(D)-C viene trascritto in un

lungo pre-mRNA che a livello nucleare viene modificato mediante taglio

degli introni e ricongiunzione (splicing) (livello c). Il meccanismo con cui

V-J e V-D-J si uniscono e` dovuto alla presenza, immediatamente dopo e

prima di ciascun segmento genico, di sequenze omologhe di 7 (eptamero)

e di 9 (nonamero) basi che risultando complementari consentiranno

l'unione V-J, V-D-J. Dodici basi dopo l'eptamero e prima del nonamero e 23

basi presenti dopo il nonamero e prima dell'eptamero rappresentano i

corrispottevi segnali di giunzione.

45

Generation of diversity (3)

• La diversita` anticorpale e` inoltre aumentata considerevolmente dal fatto

che si verificano slittamenti di alcuni nucleotidi nelle giunzioni V-D-J e V-J,

con conseguente creazione di diverse sequenze amminoacidiche. Questi

slittamenti modificano solo di 1-2 aminoacidi la regione variabile, ma

questa modificazione e` sufficiente per alterare la conformazione del

dominio V e quindi il legame Ag-Ig (livello d ).

• Inoltre si e` osservato che i geni V vanno incontro a mutazione somatica

nel corso della proliferazione cellulare indotta dall' interazione con l'

antigene molto piu` frequentemente dei geni C. Le mutazioni somatiche

(livello e) avvengono principalmente per le IgG ed IgA e sono responsabili

della modificazione dell'affinita` per l' antigene delle Ig. Questi livelli di

diversita` portano a produrre almeno 1-100 miliardi di molecole Ig diverse

partendo da un limitatissimo numero di geni.

46

47

• Flanking the V, J and D exons there are unique

sequences referred to as recombination signal

sequences (RSS), which function in recombination.

Each RSS consists of a conserved nonamer and a

conserved heptamer that are separated by either 12 or

23 base pairs. The 12bp and 23 bp spaces correspond

to one or two turns of the DNA helix.

Mechanism of DNA Rearrangements

48


• Recombination

signal sequences

(RSS)

– Nonamer

– Heptamer

– 1 or 2 turn signals

• Rag-1 and Rag-2

evoluzione

L V

2

J C

1

Lambda light chains

L V

1

J C

2

Kappa light chains

Heavy chains

L V

2

J C

2 1 1

D

Heptamer

CACAGTG

GTGTCAC

Nonamer

ACAAAAACC

TGTTTTTGG

23 bp

23 bp

Two turn

RSS 2

Nonamer

GGTTTTTGT

CCAAAAACA

Heptamer

CACTGTG

GTGACAC

12 bp

12 bp

One turn

RSS 1

49

• Recombination only occurs between a 1 turn and a 2

turn signal. In the case of the 8 light chains there is a 1

turn signal upstream of the J exon and a 2 turn signal

downstream of V8. In the case of the 6 light chains there

is a 1 turn signal downstream of the V6 gene and a 2

turn signal upstream of the J exon. In the case of the

heavy chains there are 1 turn signals on each side of the

D exon and a 2 turn signal downstream of the V gene

and a 2 turn signal upstream of the J exon. Thus, this

ensures that the correct recombination events will occur.


50


• Recombination

signal sequences

(RSS)

– Nonamer

– Heptamer


• Rag-1 and Rag-2

L V

2

J C

1

Lambda light chains

L V

1

J C

2

Kappa light chains

Heavy chains

L V

2

J C

2 1 1

D

Heptamer

CACAGTG

GTGTCAC

Nonamer

ACAAAAACC

TGTTTTTGG

23 bp

23 bp

Two turn

RSS 2

Nonamer

GGTTTTTGT

CCAAAAACA

Heptamer

CACTGTG

GTGACAC

12 bp

12 bp

One turn

RSS 1

51

• The recombination event results in the removal of the

introns between V and J in the case of the light chains or

between the V, D, and J in the case of the heavy chains.

The recombination event is catalyzed by two proteins,

Rag-1 and Rag-2. Mutations in the genes for these

proteins results in a severe combined immunodeficiency

disease (both T and B cells are deficient), since these

proteins and the RSS are involved in generating both

the B and T cell receptors for antigen.


52


• Recombination

signal sequences

(RSS)

– Nonamer

– Heptamer


• Rag-1 and Rag-2

L V

2

J C

1

Lambda light chains

L V

1

J C

2

Kappa light chains

Heavy chains

L V

2

J C

2 1 1

D

Heptamer

CACAGTG

GTGTCAC

Nonamer

ACAAAAACC

TGTTTTTGG

23 bp

23 bp

Two turn

RSS 2

Nonamer

GGTTTTTGT

CCAAAAACA

Heptamer

CACTGTG

GTGACAC

12 bp

12 bp

One turn

RSS 1

53

• An individual B cell only produces one type of light chain

and one class of heavy chain. (N.B. The one exception is

that a mature B cell can produce both mu and gamma

heavy chains but the antibody specificity is the same since

the same VDJ region is found on the mu and gamma

chains. Since any B cell has both maternal and paternal

chromosomes which code for the Ig genes there must be

some orderly way in which a cell expresses its Ig genes so

as to ensure that only one type of light chain and one class

of heavy chain is produced.

Order of Ig Gene Expression

54


+

-

Heavy chain gene

VDJ recombination

Productive rearrangement ?

Yes

No

Transcribe/translate

1st

2nd

or

Apoptosis

Progenitor B cell

Pre B cell

() Heavy chain

Paternal Maternal

55


+

-

Light chain gene

VJ recombination

Productive rearrangement ?

Yes

No

Transcribe/translate

Light chain

4 th

Apoptosis

1st -

2nd -

3rd -

4th -

or

Pre B cell

Mature B cell

+

56

• When a cell successfully rearranges a heavy chain gene, it

then begins to rearrange one of its kappa light chain genes. It

is a random event whether the maternal or paternal kappa

light chain genes are selected. If the rearrangement is

unsuccessful (i.e. it does not produce a functional kappa light

chain), then it attempts to rearrange the kappa genes on the

other chromosome. If a cell successfully rearranges a kappa

light chain gene, it will be a B cell that makes an Ig with a

kappa light chain.

Order of Ig Gene Expression Kappa light chains

57

• If a cell is unsuccessful in rearranging both of its kappa light

chain genes, it then attempts to make a lambda light chain. It

is a random event whether the maternal or paternal lambda

light chain genes are selected. If the rearrangement is

unsuccessful (i.e. it does not produce a functional lambda light

chain), then it attempts to rearrange the lambda genes on the

other chromosome. If a cell successfully rearranges a lambda

light chain gene, it will be a B cell that makes an Ig with a

lambda light chain.

Order of Ig Gene Expression Lambda light chains

58

Order of Ig Gene Expression Explains:

• Why an individual B cell only produces one

kind of Ig with one kind of heavy and one

kind of light chain

• Why an individual B cell makes only makes

antibodies of one specificity

• Why there is allelic exclusion

59

Origin of Antibody Diversity Bakground

• Antibody diversity refers to the sum total of all

the possible Ab specificities that an organism can

make. It is estimated that we can make 107 – 108

different Ab molecules. One of the major

questions in immunology has been how can we

make so many different antibody molecules.

Theories which have attempted to explain the

origin of antibody diversity fall into two major

categories.

60

Origin of Antibody Diversity History

• Definition – Diversity (repertoire) is the total of

all the Ab specificities that an organism is

capable of expressing

• Theories

– Germ line theory - This theory states that we have a

different V region gene for each possible antibody we

can make.

– Somatic mutation theory - This theory state that we

have only one or a few V region genes and the

diversity is generated by somatic mutations which

occur in these genes.

61

Origin of Antibody Diversity Current concepts

• Our current thinking is that both the germ line

and somatic mutation theories have some merit.

It is thought that antibody diversity is generated

by the following mechanisms.

1 - Large number of V genes

a) 30 lambda V genes

b) 300 kappa V genes

c) 1000 heavy chain V genes

62


2 - V-J and V-D-J joining

The region where the light chain V gene and J region or

the heavy chain V gene and D and J regions come together

is in the 3rd hypervariable region. Since it is random

which V and which J or D regions come together, there

is a lot of diversity that can be generated by V-J and V-

D-J joining.

63


3. Junctional diversity

Inaccuracies in V-J and V-D and D-J recombination.

Recombination between V-J and V-D-J is not always

perfect and additional diversity can arise by errors that

occur in the recombination event that brings the V

region next to the J or D regions or the D region next to

the J region. It is estimated that these inaccuracies can

triple the diversity generated by V-J and V-D-J joining.

The diversity generated by this mechanisms is occurring

in the 3rd hypervariable region and thus, is directly

affecting the combining site of the Ab.

64

Generation of diversity (4) Schema dei livelli che portano alla Diversita` anticorpale.

a) combinazione H-L

b) numero dei geni VH, DH, JH, VL, JL

c) meccanismi ricombinatoriali (VH x DH x JH;

VL x JL)

d) slittamenti giunzionali (VH-DH; DH-JH; VL-

JL)

e) mutazione somatica

65


• Multiple V genes

• V-J and V-D-J joining

• Junctional diversity

• N region insertions

– Amino acid

sequences not

encoded in the germ

line

D region

T G G C C C

Pro Trp

C C C C C C

V gene

T G G

D region

C C C C C C

V gene

T G G

C G G C C C

Pro Arg

C C C C C C

V gene

T G G

D region

C G C C C C

Pro Pro

66

-GTCAATG

-GTCAATG- +

Break DNA

TdT adds nucleotides

Ligate

N region insertion Intron

J D

J

J

J

D

D

D

Generation of N region insertions

67


4. N region insertion

At the junction between D and J segments there is often an

insertion of a series of nucleotides which is catalyzed by

the enzyme terminal transferase. (Terminal transferase

catalyzes the radon polymerization of nucleotides into

DNA without the need for a template. This leads to

further diversity in the 3rd hypervariable region.

68


5. Somatic mutation

There is evidence that somatic mutations are occurring in

the V gene, particularly in the place that codes for the

2nd hypervariable region. Thus, somatic mutation

probably contributes to Ab diversity to some extent.

69


6. Combinatorial association

Any individual B cell has the potential to make any one of

the possible heavy chains and any one of the possible

light chains. Thus, different combinations of heavy and

light chains within an individual B cell adds further

diversity.

70


7. Combinatorial association

Due to cross reactions between antigenic determinants of

similar structure an antibody can often react with more

than one antigenic determinant. This is termed

multispecificity. Multispecificity also contributes to Ab

diversity.

71

72

Generation of Diversity

B cell receptor (Immunoglobulin)

Heavy Light

V gene segments 1000 300

D gene segments 15 -

J gene segments 4 4

N region insertion ++ -

Junctional diversity +++ +

Somatic mutation + +

V x D x J

1000 x 15 x 4 V x J

300 x 4

Total 6 x 104 1.2 x 10

3

Combinatorial association 7.2 x 107

• Ag independent process • Clonal selection

These calculations do not take into consideration the contributions of lambda light

chains, somatic mutation junctional diversity, N region insertions or multispecificity.

73

Generation of Diversity

The process of gene rearrangement of the heavy and

light chains and the combinatorial association of

these chains occurs during B cell development and

is independent of antigen. Clones of B cells

expressing all of the possible antibody specificities

are produced during development and antigen

simply selects those clones which have the

appropriate receptor. The selected clones are then

activated, proliferate and differentiate into antibody

secreting plasma cells.

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Commutazione di classe (1)

• Per commutazione di classe si intende la capacità che hanno

i linfociti B che esprimono sulla membrana IgM ed IgD, di

esprimere Ig con diversa regione C ma con la stessa regione V

(medesimo allotipo ed idiotipi ma diverso isotipo). Alla base

della commutazione di classe ci sono una serie di

riarrangiamenti che consistono nel trasferimento delle sequenze

geniche V-D-J da C ad altre sequenze C che codificano per gli

altri isotipi di catene pesanti. Tali fenomeni coinvolgono la

ricombinazione di speciali segmenti, chiamati regioni " Switch"

(regioni S), che precedono di circa 2-3 kbp ogni gene CH

eccetto C . Ciascun gene CH é costituito da tanti esoni quanti

sono i singoli domini che costituiscono le porzioni costanti delle

catene pesanti (3 per IgG, IgA, IgD e 4 per IgM ed IgE. Tra gli

esoni CH1 e CH2 dei geni C sono presenti delle porzioni geniche

H (hinge) che codificano per la regione cerniera.

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Commutazione di classe (2)

Sono stati proposti due meccanismi molecolari per spiegare la

commutazione di classe e quindi la ricombinazione S-S. Tale

evento infatti può avvenire o in seguito a delezione di

materiale genico e quindi essere un evento unidirezionale ed

irreversibile o con scambio tra cromatidi fratelli e quindi

introduzione di più copie di geni C senza eliminazione di

materiale genetico. Inoltre, le interazioni specifiche tra

differenti regioni S possono essere attivate da proteine che

riconoscono sequenze specifiche delle regioni S.

Ogni singolo clone linfocitario può commutare più volte fino

all'espressione di IgA, il cui gene é ultimo nella regione C

(organizzazione genica del topo).

Il fenomeno della commutazione di classe è regolato da fattori

solubili prodotti dai linfociti T (citochine).

76

77

78

79

Commutazione Ig-membrana - Ig-secretoria (Igm-Igs).

Le Ig di membrana sono ancorate alla cellula grazie alla

presenza nella porzione carbossiterminale di 41 aa idrofobici.

Tale porzione è codificata da due piccoli esoni (M1 e M2) che

giacciono al di là degli esoni che codificano per la porzione CH.

Quando un linfocita B in seguito alla interazione con l'antigene

inizia a secernere Ig, avviene un fenomeno di "switch" che

coinvolge la processazione del RNA trascritto. Infatti la

trascrizione dell'RNA termina prima degli esoni M1 e M2 ed

una piccola porzione (S) dell' ultimo esone CH regola la

porzione carbossiterminale. Quindi se la trascrizione si arresta

in corrispondenza di S, si ottiene una Ig secretoria, mentre se

la trascrizione procede includendo gli esoni M1 e M2, la

saldatura rimuove S e si avrà una Ig di membrana. Nella

plasmacellula esiste un meccanismo in grado di far bloccare la

trascrizione prima di M1 e M2 e così tutte le Ig saranno di

tipo secretorio.

80

I linfociti risultano dotati di doppi cromosomi (alleli), uno di

origine paterna ed uno di origine materna. Quindi nel corso

del differenziamento, ogni linfocita dovra` scegliere non solo

quale catena kappa o lambda (esclusione isotipica) ma

anche quale allele utilizzare (esclusione allelica). Questi

due fenomeni di esclusione comportano che ogni linfocita B

debba scegliere uno dei 4 geni L ed uno dei 2 H. Durante lo

sviluppo prima viene attivato un gene H (quindi si troveranno

solo catene H intracitoplasmatiche, linfocita pre-B) .

Esclusione allelica ed esclusione isotipica (1)

81

Avvenuta l'espressione del gene H, avviene il riarrangiamento

dei geni L, in particolare prima riarrangiano i geni k, ed

appena il riarrangiamento é funzionale, cessa ogni altro tipo

di riarrangiamento a carico dei geni L. Le catene M e k si

assemblano e muovono verso la membrana dove si

ancorano. Se invece i due loci falliscono, i geni

riarrangiano e si avranno Ig di membrana

Esclusione allelica ed esclusione isotipica (2)

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• A cell first attempts to rearrange one of its heavy chain genes;

in some cells the maternal chromosome is selected and in

others the paternal chromosome is selected. If the

rearrangement is successful so that a heavy chain is made,

then no further rearrangements occur in the heavy chain

genes. If, on the other hand, the first attempt to rearrange the

heavy chain genes is unsuccessful (i.e. no heavy chain is

made), then the cell attempts to rearrange the heavy chain

genes on its other chromosome. If the cell is unsuccessful in

rearranging the heavy chain genes the second time, it is

destined to be eliminated.

Order of Ig Gene Expression Heavy chains

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Corso di Immunologia A.A. 2008-09 Genetica …