Advanced Immunology, Dr. Aguilera 10/7/2010utminers.utep.edu/raguilera/10-UTEP-Immunollec.pdf · Advanced Immunology, Dr. Aguilera 10/7/2010 5 Because of allelic exclusion, the Ig

Advanced Immunology, Dr. Aguilera 10/7/2010

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Chapter 5

Organization and Expression ofOrganization and Expression of

Immunoglobulin GenesImmunoglobulin Genes

Bone MarrowBone Marrow

TT--CellsCells Lymph nodesLymph nodes

SpleenSpleen

ThymusThymus

}} T+BT+B--cellscells

HSCs and BHSCs and B--cellscells

BB--lymphocytes produce antibodieslymphocytes produce antibodies

{Antigen Binding Variable Region

Constant RegionDomain

Immunoglobulin (Ig) MoleculeImmunoglobulin (Ig) Molecule

{

Heavy-chain

see Fig. 4.6

Light-chain

•• Theoretically, antibodies (Abs) can beTheoretically, antibodies (Abs) can be

produced to just about any foreign produced to just about any foreign

substance and are highly specificsubstance and are highly specific

•• So we would need millions of Abs to do thisSo we would need millions of Abs to do this

An antibody can distinguish one proteinAn antibody can distinguish one protein

from another by a single amino acid from another by a single amino acid differencedifference

Ex.Ex.


2

•• There were two main hypotheses for the origin of this diversity. There were two main hypotheses for the origin of this diversity.

•• 1) 1) GermlineGermline theorytheory: :

--Held that there is a separate gene for each different Held that there is a separate gene for each different

immunoglobulin chain and that the antibody repertoire is largely immunoglobulin chain and that the antibody repertoire is largely

inherited. inherited.

•• 2) 2) Somatic diversification theorySomatic diversification theory: :

--Proposed that the observed repertoire is generated from a Proposed that the observed repertoire is generated from a

limited number of inherited variable (V)limited number of inherited variable (V)--region sequences that region sequences that

undergo alteration within B cells during the individual's lifetime. undergo alteration within B cells during the individual's lifetime.

Clonal Selection Hypothesis:Clonal Selection Hypothesis: An individual cell expressesAn individual cell expressesa specific receptor that recognizes a unique antigena specific receptor that recognizes a unique antigen--specificity determined prior to the presence of antigenspecificity determined prior to the presence of antigen

Binding of antigen to receptor induces proliferation withBinding of antigen to receptor induces proliferation witheach daughter cell producing the same antibody each daughter cell producing the same antibody specificity (to the original activating antigen)specificity (to the original activating antigen)

Specific Antigen

To produce the millions of differentTo produce the millions of different

antibodies necessary to combat disease,antibodies necessary to combat disease,

millions of antibody genes must have millions of antibody genes must have evolved to encode this informationevolved to encode this information

Ig receptors genes did not follow 1Ig receptors genes did not follow 1--gene/1gene/1--protein theoryprotein theory

Since one gene encodes one proteinSince one gene encodes one protein

(generally), this would mean that cells (generally), this would mean that cells

would need more genes than potentially would need more genes than potentially encoded by genome encoded by genome

1987 Nobel Prize1987 Nobel Prize

Susumu TonegawaSusumu Tonegawa

Using lightUsing light--chain mRNA as probes was able to chain mRNA as probes was able to demonstrate that the variable region and the constant demonstrate that the variable region and the constant

regions were “rearranged” in Bregions were “rearranged” in B--cell tumors (plasmacytomas)cell tumors (plasmacytomas)

The answer to this problem resulted in a Nobel Prize The answer to this problem resulted in a Nobel Prize

germlinealleles

rearranged alleles

Liver B-cell tumor

identical alleles rearranged alleles

Southern Analysis of Immunoglobulin Gene Alleles

See Fig. 5.2

V J

VV--(D)(D)--J (Joining) RecombinationJ (Joining) Recombination

JV D J23 bp-RSS 23 bp-RSS

V J CµEnh

12 bp-RSS12 bp-RSS

VDJCµµµµ mRNA

~~~~~~~~~~~~~~~~~~~~~~~~

heavyheavy--chainchain

~23 648

see Fig. 5.5


3

Joining-element coding region

CACAGTGCACAGTG7mer7mer

Recombination Signal Sequences (RSS)Recombination Signal Sequences (RSS)

23 bp

V and J gene segments contain same recombination elements

ACAAAAACCACAAAAACC9mer9mer

See Fig. 5.6

HEPTAMERHEPTAMER NONAMERNONAMER

ACAAAAACCCACAGTG

12 bp RSS12 bp RSS

23 bp RSS23 bp RSS

ONEONE--TURNTURN

TWOTWO--TURNTURN

The 12bp/23bp Spacer Rule Regulates V-D-J joining

23bp23bp--SPACERSPACER

ACAAAAACCCACAGTG 12bp12bp--SPACERSPACER

See Fig. 5.6

Specific signals are necessary toSpecific signals are necessary toensure V to J and prevent ensure V to J and prevent V to V and J to J recombinationV to V and J to J recombination

Signals are highly evolutionarily Signals are highly evolutionarily conservedconserved--from sharks to manfrom sharks to man See Fig. 5.6

DeletionDeletion

InversionInversion

V J

DeletedDeletedCircleCircle InvertedInverted

DNADNA

VJVJVJVJ

77--1212--9999--2323--77

Recombination can proceed via deletion or inversion

See Fig. 5.7

Junctional flexibility

Fig. 5.9

Experimental evidence for junctional flexibility inimmunoglobulin-gene rearrangementRSS= Recombination signal-sequence flanking each germline V, D, and J genesegments.

Junctional flexibility

Fig. 5.12


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•• Seven means of antibody diversification have been Seven means of antibody diversification have been identified in mice and humans:identified in mice and humans:

1.1. Multiple germMultiple germ--line gene segmentsline gene segments

2.2. Combinatorial VCombinatorial V--(D)(D)--JJ--joiningjoining

3.3. JunctionalJunctional flexibilityflexibility

4.4. nucleotide addition (at junctions)nucleotide addition (at junctions)

5.5. Somatic Somatic hypermutationhypermutation

6.6. Combinatorial association of light and heavy chainsCombinatorial association of light and heavy chains

In humans, potential antibody combining-site diversityover 1010 (10 billion)

“Recombinase” factors recognize the Recombination Sequences (RSS)

Deleted DNA

Recombinase complex

Pre-B

Recombinase is expressed early during B-Lymphocyte differentiation

Variable Region Formation

stem cell

VDJ and VJ RecombinationVDJ and VJ Recombinationoccur only in pro/preoccur only in pro/pre--B cell stageB cell stage

B-cell Plasma-B

IgM

IgD

Antigen Independent StagesAntigen Independent Stages

LymphocyteLymphocyte--Specific Enzymes Specific Enzymes Simultaneous Cleavage at both RSSs Simultaneous Cleavage at both RSSs

See Fig. 5.7

RecombinaseRecombinase Activating Genes (RAGs)Activating Genes (RAGs)

These data show that the These data show that the

frequency of mutation:frequency of mutation:

(1)(1)increases in the course of the increases in the course of the

primary response (day 7 vs. primary response (day 7 vs.

day 14);day 14);

(2) Is higher after secondary and (2) Is higher after secondary and

tertiary immunizations than after tertiary immunizations than after

primary immunization.primary immunization.

(3) Only happens at Variable (3) Only happens at Variable

regions and not at any other site regions and not at any other site

or gene (too or gene (too dangerorsdangerors))

Somatic Hypermutation & Affinity Maturation)

Affinity increases over time


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Because of allelic exclusion, the Because of allelic exclusion, the

IgIg heavyheavy-- and lightand light--chain genes of chain genes of

only one parental chromosome only one parental chromosome

are expressed per cell.are expressed per cell.

This process ensures that B cells This process ensures that B cells

possess a single antigenic possess a single antigenic

specificity.specificity.

The allele selected for The allele selected for

rearrangement is chosen rearrangement is chosen

randomlyrandomly..

Thus, Thus, the expressed the expressed IgIg may may

contain one maternal and one contain one maternal and one

paternal chain or both chains may paternal chain or both chains may

derive from only one parentderive from only one parent. .

Only lymphocytes do thisOnly lymphocytes do this

* Expressed alleles

Allelic ExclusionAllelic Exclusion

• All the progeny of that B cell will express the same assembled V genes.

• Every B cell begins by expressing IgM as its B-cell receptor, and the first antibody produced in an immune response is always IgM.

• Later in the immune response, however, the same assembled V region may be expressed in IgG, IgA, or IgE antibodies.

• This change is known as isotype switching.

Isotype or Class Switching

There are several antibody isotypes There are several antibody isotypes

Fig. 3.20

highest lowest

��Different IgDifferent Ig isotypesisotypes are needed for surveillanceare needed for surveillance

at different sites in the bodyat different sites in the body

IgIg HeavyHeavy--Chain Chain isotypeisotype switch recombinationswitch recombination

V JD CµµµµSµµµµ

SααααSµµµµ

Sγγγγ1 Cγγγγ1 CααααSαααα

CααααV JD

Factors (AID)

change effector function without changing specificitychange effector function without changing specificity

~500 kb away

EnhEnh

E

3’Enh3’Enh

Proposed mechanism for class switching in rearranged Ig heavy-chain genes.

A switch (S) site is located upstream from each CH segment except Cδδδδ.

.

Cytokines like IL-4 stimulate switching via AID

• Switching to other isotypes occurs only after B cells are

stimulated in the course of an immune response by external

signals, such as cytokines released by T cells or mitogenic signals

delivered by pathogens.

• It occurs through a specialized DNA recombination mechanism

guided by stretches of repetitive DNA known as switch regions.

• Switch regions lie in the intron between the JH gene segments and

the Cµ gene, and at equivalent sites upstream of the genes for

each of the other heavy-chain isotypes, with the exception of the δ

gene.

• The µ switch region (Sµ) consists of G-rich repetitive sequences

Isotype or Class Switching


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AID (activationAID (activation--induced induced cytidinecytidine deaminasedeaminase) mediates ) mediates both somatic both somatic hypermutationhypermutation and class switchingand class switching

•• Class switching depends on the interplay of 4 elements:Class switching depends on the interplay of 4 elements:

1.1. switch (S) regionsswitch (S) regions2.2. the cytokine signals the cytokine signals 3.3. enzyme enzyme activationactivation--induced induced cytidinecytidine deaminasedeaminase

(AID) + accessory factors(AID) + accessory factors

•• AIDAID is theis the key mediator of somatic key mediator of somatic hypermutationhypermutation and and classclass--switch recombinationswitch recombination..

•• It belongs to the It belongs to the family of RNAfamily of RNA--editing enzymesediting enzymes..

•• AID AID deaminatesdeaminates selected selected cytosinescytosines in certain mRNAs, in certain mRNAs, changing cytosine to changing cytosine to uracilsuracils, thereby altering (editing) the , thereby altering (editing) the proteinprotein--encoding instructions of the targeted mRNA.encoding instructions of the targeted mRNA.

AID (activation-induced cytidine deaminase) mediates both

somatic hypermutation and class switching

AID mutant mice do not switch to IgG and do not undergo somatic hypermutation.

Expression of secreted and membrane

forms of µµµµ and δδδδ the heavy chain by

alternative RNA processing.

(a) Structure of the rearranged heavy-chain

gene showing the Cµ exons and Cδ exons

and poly-A sites.

(b) Structure of µm transcript and µm mRNA

resulting from polyadenylation at site 2 and

splicing.

(c) Structure of δm transcript and δm mRNA

resulting from polyadenylation at site 4 and

splicing.

Both processing pathways can proceed

in any given B cell

Fig. 5.19.

a

b

c

Regulation of Ig-gene transcription

•• TwoTwo majormajor ciscis regulatoryregulatory sequencessequences inin DNADNA regulateregulate transcriptiontranscription ofof IgIg genesgenes::

•• PromotersPromoters::

--RelativelyRelatively shortshort sequencessequences nearnear thethe transcriptiontranscription initiationinitiation sitesite thatthat promotepromote

initiationinitiation ofof RNARNA transcriptiontranscription inin aa specificspecific directiondirection..

•• TheThe promoterpromoter regionregion isis recognizedrecognized byby RNARNA Polymerase,Polymerase, whichwhich thenthen initiatesinitiates

transcriptiontranscription..

•• PromotersPromoters controlcontrol whichwhich genesgenes areare transcribedtranscribed (cell(cell--specificspecific oror not)not) andand whatwhat

proteinsproteins thethe cellcell manufacturesmanufactures..

•• Enhancers:Enhancers:

--DNA Regions situated some distance upstream or downstream from a gene that DNA Regions situated some distance upstream or downstream from a gene that

activate transcription from the promoter sequence in an orientationactivate transcription from the promoter sequence in an orientation--independent independent

manner (most are cellmanner (most are cell--type or tissue specific).type or tissue specific).

Regulation of Ig-gene transcription

Location of promoters (dark red) and enhancers(green) in mouse heavy-chain, kappa light chain, andlambda light-chain.

Advanced Immunology, Dr. Aguilera 10/7/2010utminers.utep.edu/raguilera/10-UTEP-Immunollec.pdf · Advanced Immunology, Dr. Aguilera 10/7/2010 5 Because of allelic exclusion, the Ig

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