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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.
Advanced Immunology, Dr. Aguilera 10/7/2010
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•• 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
Advanced Immunology, Dr. Aguilera 10/7/2010
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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
Advanced Immunology, Dr. Aguilera 10/7/2010
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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
Advanced Immunology, Dr. Aguilera 10/7/2010
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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.