Lecture 3 - University of Texas at El Paso

Advanced Immunology, Dr. Aguilera 2014

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Lecture 3

VV--Gene Rearrangement and ExpressionGene Rearrangement and Expression

Rat anti–mouse CD19, anti–mouse B220, and anti–mouse CD22. Mice were then injected with a secondary antibody mouse anti–rat antibody

Used anti –B cell marker antibodies to deplete in mice


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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


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{Antigen Binding Variable Region

Constant RegionDomain

Immunoglobulin (Ig) MoleculeImmunoglobulin (Ig) Molecule

{Heavy-chain

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

Ex.Ex.


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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


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To produce the millions of differentTo produce the millions of differentantibodies 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

IgIg receptors genes did not follow 1receptors genes did not follow 1--gene/1gene/1--protein ruleprotein rule

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


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germlinealleles

rearranged alleles

Liver B-cell tumor

identical alleles rearranged alleles

Southern Analysis of Immunoglobulin Gene Alleles

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


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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

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


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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

DeletionDeletion

InversionInversion

V J

DeletedDeletedCircleCircle InvertedInverted

DNADNA

VJVJVJVJ

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

Recombination can proceed via deletion or inversion


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Junctional flexibility

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

segments.

Junctional flexibility


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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)


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Primordial Receptor Gene

V D J C

light-chain

heavy-chainκ λκ λκ λκ λ

α β γ δα β γ δα β γ δα β γ δ

T-Cell AntigenReceptor

MHC

CD4 CD8 Thy-1

recognized self vs non-self?

Antibody

Immunoglobulin gene family

T-cell Antigen Receptors Resemble Antibody molecules


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TT--cell Antigen Receptor Genescell Antigen Receptor Genes

TT--cell antigen receptor also undergoes cell antigen receptor also undergoes SiteSite--specific rearrangementspecific rearrangement

using the same recombination signalsusing the same recombination signals

TCRTCR--ββββββββ genesgenes

TCRTCR--α/δα/δα/δα/δα/δα/δα/δα/δ genesgenes

TCRTCR--γ γ γ γ γ γ γ γ genesgenes

TCR δδδδ loci


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What factors might be involved in VDJ Recombination?

Sequence-Specific DNA Binding Factors?

Site-Specific Cleavage Activity?

DNA Ligase Activity?

Other "House-keeping" Factors (DNA Repair)?

What are the “Recombinase” factors that recognize the Recombination Sequences (RSS)?

Deleted DNA

recombinase


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�Pre-B cell lines were created by Abelson Murine Leukemiatransformation of mouse bone marrow cells in vivo+in vitro

The Search for the Recombinase ComplexThe Search for the Recombinase Complex

�In early 1980s, retroviral vectors were developed in the laboratory of David Baltimore to study the mechanism of V-(D)-J recombination

�These retroviral vectors revealed that V-(D)-J recombinationcan be reproduced in the Pre-B cell lines, but not other cells

�These results lead to a frantic search for the V(D)J recombinase

Retroviral Recombination Reporter VectorsRetroviral Recombination Reporter Vectors

LTR

gpt

LTR

V

J

LTR gpt

V J

LTR

Rearrangement by Inversion Confers Drug ResistanceRearrangement by Inversion Confers Drug Resistance

genomic

DNA

gpt = xanthine-guanine phosphoribosyltransferase gene


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Pre-B

Recombinase must be expressed early during B-Lymphocyte differentiation

Variable Region Formation

stem cell

B-cell Plasma-B

Retroviral vectors were found toRetroviral vectors were found torearrange only in pro/prerearrange only in pro/pre--B cell linesB cell lines

but not in nonbut not in non--lymphoid or mature B cellslymphoid or mature B cells

““Chance Favors a Prepared MindChance Favors a Prepared Mind””Louis Pasteur


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�A student performs a series of flawed experiments that

leads to the discovery of the V-(D)-J recombinase

An improbable experiment leads to an “incredible” resultAn improbable experiment leads to an “incredible” result

This experiment should never have worked!This experiment should never have worked!Why?Why?

�The premise of the experiment was that a single recombinase gene was responsible for V-(D)-J joining

�Reasoned that a recombinase gene could be transferred from lymphocyte DNA to a cell that does not contain this activitysuch as fibroblasts

LymphocyteLymphocyte--specific genes should not be expressed specific genes should not be expressed in nonin non--lymphoid cells lymphoid cells -- also unreasonable to believealso unreasonable to believe

that one gene product could do everythingthat one gene product could do everything

David Schatz, 2001


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LTR

Fig. 1 Schatz and Baltimore (1987)

LTRgpt

VJa Jb

LTR

probe

gptLTR

untranscribed gpt gene

Ja JbVκ

inversion

Retroviral vectors used to detect siteRetroviral vectors used to detect site--specific recombination (inversion)specific recombination (inversion)

Inversion allowsInversion allows

expression of expression of

sense gpt genesense gpt gene

Table I. Table I.

Developmental Stage Specificity of V(D)J Recombination ActivityDevelopmental Stage Specificity of V(D)J Recombination Activity

Cell type Growth under Selection Rec. Freq.

(recombination) (event/cell div.)

Lymphocyte precursor None <1/2x106

(Ba/F3 cell line)

PrePre--B cellsB cells YESYES 1/1x101/1x1033

(38B9, PD31)

B-Cell (IgM Positive) None <1/4x106

(Wehi 231)

Fibroblast None <1/2x107

(NIH 3T3)

As expected, PreAs expected, Pre--B cell lines contain a specific recombinase activityB cell lines contain a specific recombinase activity

Schatz and Baltimore (1987)


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Sheared Human DNASheared Human DNA Retroviral VRetroviral V--DD--J J Reporter construct Reporter construct

mouse fibroblast

(no recombinase activity)

Isolate Human GeneIsolate Human GeneResponsible for Recombination ActivityResponsible for Recombination Activity

select with specific drugselect with specific drug

The Search for the Recombinase GeneThe Search for the Recombinase Gene

only drug resistant clonesonly drug resistant clonesunderwent recombinationunderwent recombination

Genomic DNA + pSV2Genomic DNA + pSV2--His (histidinol dehydrogenase)His (histidinol dehydrogenase)

Select for recombinationSelect for recombination

by gpt activationby gpt activation

with mycophenolic acidwith mycophenolic acid

A few gptA few gptrr clones were obtainedclones were obtained

Select for Histidinol resistanceSelect for Histidinol resistance

Select for uptake of foreign DNASelect for uptake of foreign DNA


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unrearrangedunrearranged

Inverted Inverted rearrangedrearrangedconstructsconstructs

VV--JaJa

Southern analysis of drug resistant fibroblasts contain Southern analysis of drug resistant fibroblasts contain expected recombination eventsexpected recombination events

controls gptr clones


TR-1

+ -

gpt Ja JbVκ

gpt

RSSs

VκJa


Rearrangements are consistent with siteRearrangements are consistent with site--specific Vspecific V--J joiningJ joining

Vκκκκ JκκκκGGATCCT* *

GGATCCTCC

TR-1

TRX-1 **GGACGTTCGGTGGAGGCAC

**GGACGTTCGGTGGAGGCAC

*deleted bp


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Identification of VIdentification of V--(D)(D)--J Recombinase Gene J Recombinase Gene

3TGR cells (Fibroblasts w/ LTR Recombination Vector)

Human DNAHuman DNA

Stable Transfer of Recombination Activity

What gene caused this effect?What gene caused this effect?

Link oligos to genomicLink oligos to genomic

DNA after RE digestionDNA after RE digestion

How do you isolate a human gene within a sea of mouse and human genes?How do you isolate a human gene within a sea of mouse and human genes?

different sized fragments on 3’ sidedifferent sized fragments on 3’ side

Fig. 2Schatz et al., 1989

2°°°° transfectants

1°°°° transfectant


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Original Original RAGRAG--11 Germline Clone Contained Another GeneGermline Clone Contained Another Gene

RAGRAG--11 RAGRAG--22

Fig.1 Oettinger, et al., 1990

Probe J detected2.2 kb mRNA by

Northern in pre-B cells

Fibroblasts

High level recombinationHigh level recombination

RAG-1 cDNA

RAG-2 cDNA

RAGRAG--22 and and RAGRAG--11 are sufficient for high level recombination are sufficient for high level recombination


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Cell Oligo+

line DNA Ampr CamrAmpr R

3TGR 0 184,000 0 0

3TGR RAG-1 60,400 0 0

3TGR RAG-2 50,000 0 0

3TGR RAG-1 + RAG-2 70,600 490 0.7

NIH 3T3 RAG-1 + RAG-2 193,600 2,166 1.1

NIH 3T3 Human RAG-1 + 73,200 372 0.5mouse RAG-2

*Total number of independent transfections

Efficient Rearrangement only found when bothEfficient Rearrangement only found when bothRAGRAG--11 and and RAGRAG--22 were cotransfected into fibroblastswere cotransfected into fibroblasts

Table I RAGRAG--11 and and RAGRAG--22 Act SynergisticallyAct Synergistically

Oettinger, et al., 1990

RAGRAG--11

RAGRAG--22

Mature B Mature T

Pre-T

Pro/Pre-B

Fibroblast

Expression of the Expression of the RAGRAG--22 is Pro/Preis Pro/Pre--Lymphocyte SpecificLymphocyte Specific

Fig.4


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Targeted Disruption of Targeted Disruption of RAGRAG--11 and and RAGRAG--22 yieldsyieldsconclusive proof that the RAG genes are essentialconclusive proof that the RAG genes are essential

for Vfor V--(D)(D)--J recombinationJ recombination

Shinkai, Y., et al., (1992).RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J recombination. Cell 68:855-867.

Mombaerts, P., et al., (1992).RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68:869-877.

Generation of Generation of RAGRAG--1/21/2 knockoutsknockouts

Homozygous neor Embryonic Stem Cells (ES)

wt RAG gene

Disrupted gene

neo


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Lymphocyte surface “markers”Lymphocyte surface “markers”Used for isolation and identification Used for isolation and identification

(Tyr-PPase)

B220

SpleenSpleen

Wild-type RAG-2 Knockout

55%0%

Surface IgM

FACS Analysis of Lymphocyte PopulationsFACS Analysis of Lymphocyte Populations

double positiveB220+/IgM+

(mature B-cells)

B220

Surface IgM

BoneBone

marrowmarrow26%

0%

Pro/Pre-B


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αβαβαβαβ

CD3

Wild-type RAG-1 mutant

ThymusThymus

0%T-cellantigenreceptor

CD4

CD8

No mature TNo mature T--cellscells

Thy-1

IL-2R Precursor TPrecursor T--cellscells

No functional TNo functional T--cellscells

RAG RAG --//-- “knockout” Mutant Mice“knockout” Mutant Mice

�� Normal birth and growth but immunodeficient

�� Have no Mature B/T cells

�� No V-(D)-J recombination

�� Higher levels of precursor-T cells (Thy-1+, IL-2R+)

�� Higher levels of NK+, macrophages, and granulocytesprobably filling a “void” left by loss of lymphocytes


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Important questions:Important questions:

�� Do the RAGs bind and cut the RSS?Do the RAGs bind and cut the RSS?

�� Can a recombination system be established orCan a recombination system be established ordissected dissected in vitroin vitro??

Characterization of the Recombination Mechanism Characterization of the Recombination Mechanism in vitroin vitro

van Gent, D.C. et al.,. (1995). Initiation of V(D)J recombination in a

cell-free system. Cell, 81:925-934

Martin Gellert’s Laboratory at NIH makes initial and consistentMartin Gellert’s Laboratory at NIH makes initial and consistent

discoveries which lead to the establishment of discoveries which lead to the establishment of in vitroin vitro rec. systemsrec. systems


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Other important findings:

�� RAG mediated cleavage requires intact RSS heptamer and nonamer sequences

�� Cleavage products were identical to those detected in vivo-

blunt 5’ phosphorylated signal-ends and coding-ends containhairpins (closed covalently)

�� Recombinant Rag-1 and Rag-2 proteins plus house-keeping

proteins are sufficient to mediate recombination in vitro

RAG Mediated Cleavage

nick

Transesterification reaction

RAG-1RAG-232P

nick

hairpin

-RSS

hairpin


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�Recombinant Rag-1 and Rag-2 were subsequentlyshown to mediate all the cleavage steps in vitro

Can the Can the in vitroin vitro system perform all the steps seen system perform all the steps seen in vivoin vivo??

These results lead to the following question:These results lead to the following question:

�Rag-1 and Rag-2 forms a large stable cleavage complex that requires an intact heptamer and nonamer

Initiation of V(D)J recombination Initiation of V(D)J recombination in vitroin vitro obeying the 12/23 ruleobeying the 12/23 rule

Eastman, Q.E., Leu, T.M, and Schatz, D. G. Nature, 380:85-88, 1996

time of reaction

12bp-RSS 23bp-RSS

12bp-RSS

23bp-RSS

For efficient cleavage, needed RAGFor efficient cleavage, needed RAG--1+RAG1+RAG--2 2 + Nuclear Extract (additional factors)+ Nuclear Extract (additional factors)


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1/12/05 Advanced Immunology Dr. Aguilera

B/T Lymphocytes

RAGs and V-(D)-J recombination appeared early in vertebrate evolution

450 x 106 years ago

Big-Bang Theory of Immunology

Primitive Receptor Gene

Exon 1 Exon 2

Transposable elements created the first antigen receptor genes

RAG Transposon


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Furthermore, the RAG genes had to come under the control of lymphocyte-specific promoters

lymphocyte-specificpromoter

The RAG genes become part of host’s genome

defective or absent RSS

synapsissynapsis

Simultaneous Cleavage at both RSSs Simultaneous Cleavage at both RSSs

Housekeeping Housekeeping enzymesenzymes

ligate and repair ligate and repair breaksbreaks

Mg++


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Malu el al 2012

DNA repairDNA repairenzymes enzymes are also are also needed needed

RAGs are more complex than anyone thought


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Control of recombination is complicated

Lecture 3 - University of Texas at El Paso

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