Immunology Lecture 1, Dr. Aguilera 2/9/2009

Immunology Lecture 1, Dr. Aguilera 2/9/2009

Lecture 1

Lymphocytes and Antigen ReceptorsLymphocytes and Antigen Receptors

posted at http://posted at http://utminers.utep.edu/raguilerautminers.utep.edu/raguileraB/T Lymphocytes

450 million years ago

All organisms have some sort of immune system

Insects also have a sophisticated native immunesystem composed of specialized cells and hundreds

of antimicrobial response peptides/proteins

Self-non-self recognition

HSCCommitted Lymphocyte Precursor

Nat. Rev. Immunol.

CLP

T-cells

NK cells

B-cells

All lymphocytes arise from a common precursor

BB--lymphocytes produce antibodieslymphocytes produce antibodies


{Antigen Binding Variable Region

Immunoglobulin (Immunoglobulin (IgIg) Molecule) Molecule

{

{

Constant RegionDomain

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

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.

1930s1930s--50s50s--Many theories were proposed to explain howMany theories were proposed to explain how

so many different antibodies can be made to so many different antibodies can be made to

a a ““UniverseUniverse”” of different antigensof different antigens

Theories used to explain antibody diversityTheories used to explain antibody diversity

original shapeoriginal shape modified shapemodified shape

Instructional TheoryInstructional Theory:: Antigens would serve as moldsAntigens would serve as molds

to to ““instructinstruct”” the creation of a specific antibodythe creation of a specific antibody

Selective TheorySelective Theory:: Single cells express a large number of Single cells express a large number of

receptors that upon contact with antigen would inducereceptors that upon contact with antigen would induce

the cell to release more of the the cell to release more of the ““selectedselected”” receptorreceptor


ClonalClonal Selection Hypothesis:Selection Hypothesis: An individual cell expressesAn individual cell expresses

a 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

Correct HypothesisCorrect Hypothesis::

Binding of antigen to receptor induces proliferation withBinding of antigen to receptor induces proliferation with

each daughter cell producing the same antibody each daughter cell producing the same antibody

specificity (to activating antigen)specificity (to activating antigen)

Specific Antigen

To produce the billions of differentTo produce the billions of different

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

billions of antibody genes must have billions 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 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 Susumu TonegawaTonegawa

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 regions were ““rearrangedrearranged”” in Bin B--cell tumors (cell tumors (plasmacytomasplasmacytomas))

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

germline

allelesrearranged alleles

Liver B-cell

identical alleles rearranged alleles

Southern Analysis of Immunoglobulin Gene Alleles


V J

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

JV D J23 bp-RSS 23 bp-RSS

V J CµEnh

12 bp-RSS12 bp-RSS

VDJCµµµµ mRNA

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

heavyheavy--chainchain

~15 4~ 100s

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 12 bpbp RSSRSS

23 23 bpbp RSSRSS

ONEONE--TURNTURN

TWOTWO--TURNTURN

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

23bp23bp--SPACERSPACER

ACAAAAACCCACAGTG 12bp12bp--SPACERSPACER

Specific signals are necessary toSpecific signals are necessary to

ensure 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

DeletedDeleted

CircleCircle InvertedInverted

DNADNA

VJVJVJVJ

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

Recombination can proceed via deletion or inversion T-cell Antigen Receptors Resemble Antibodies

TT--cell Antigen Receptor Genescell Antigen Receptor Genes

TT--lymphocytes express an antigen receptor lymphocytes express an antigen receptor

that also undergoes site specific rearrangementthat also undergoes site specific rearrangement

using exactly the same recombination signalsusing exactly 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

Diversity of Antigen Receptor Genes

Form Form HeterodimersHeterodimers (Ig H+L and TCRαβαβαβαβ & TCRγδγδγδγδ))))

Random Gene AssortmentRandom Gene Assortment

Flexible RecombinationFlexible RecombinationAddition and Deletion of bases at junctions+ reading frame changes

Somatic Somatic HypermutationHypermutation (B(B--cells)cells)Alteration the rearranged V genesgenerally confined to hypervariable regionsleads to “selection” of higher affinity antibodies


Rearrangement proceeds in an ordered fashion during B-cell Differentiation

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


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

The Search for the The Search for the RecombinaseRecombinase ComplexComplex

�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

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 to

rearrange 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


�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 An improbable experiment leads to an ““incredibleincredible”” resultresult

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

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 sense gptgpt genegene

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)


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

Reporter construct Reporter construct

mouse fibroblast (no recombinase activity)

Isolate Human GeneIsolate Human Gene

Responsible for Recombination ActivityResponsible for Recombination Activity

select with specific drugselect with specific drug

The Search for the The Search for the RecombinaseRecombinase GeneGene

LTRgpt

LTR

only drug resistant clonesonly drug resistant clones

underwent recombinationunderwent recombination

Genomic DNA + pSV2Genomic DNA + pSV2--His (His (histidinolhistidinol dehydrogenasedehydrogenase))

Select for recombinationSelect for recombination

by by gptgpt activationactivation

with with mycophenolicmycophenolic acidacid

A few A few gptgptrr clones were obtainedclones were obtained

Select for Select for HistidinolHistidinol resistanceresistance

Select for uptake of foreign DNASelect for uptake of foreign DNA

unrearrangedunrearranged

Inverted Inverted

rearrangedrearranged

constructsconstructs

VV--JaJa

Southern analysis of drug resistant fibroblasts contain Southern analysis of drug resistant fibroblasts contain

expected recombination eventsexpected recombination events

controls gptr clones


TRX-1TR-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-1TRX-1 & 2

**GGACGTTCGGTGGAGGCAC

**GGACGTTCGGTGGAGGCAC

*deleted bp


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 Link oligosoligos to genomicto genomic

DNA after RE digestionDNA after RE digestion

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

different sized fragments on 3different sized fragments on 3’’ sideside

Fig. 2Schatz et al., 1989

2°°°° transfectants

1°°°° transfectant

Original Original RAGRAG--11 GermlineGermline Clone Contained Another GeneClone 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


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 both

RAGRAG--11 and and RAGRAG--22 were were cotransfectedcotransfected into fibroblastsinto fibroblasts

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

Oettinger, et al., 1990

RAGRAG--11

RAGRAG--22

actin

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

Targeted Disruption of Targeted Disruption of RAGRAG--11 and and RAGRAG--22 yieldsyields

conclusive 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


LymphocyteLymphocyte--cell surface receptors serve as cell cell surface receptors serve as cell ““markersmarkers””

which are needed for their isolation and identification which are needed for their isolation and identification

(Tyr-PPase)

FluorescenceFluorescence

Activated CellActivated Cell--SorterSorter

Collect or analyzesorted cells

Laser beam

Input cells

Labeled antibodies against cellLabeled antibodies against cell

surface markers are usedsurface markers are used

to isolate cell populationsto isolate cell populations

FACS or Flow FACS or Flow CytomeryCytomery

FITC, Rhodamine,Texas Red, etc

Flow Cytometer and Sorter EPICS ALTRA

Post-Sort cellsPre-Sort cellsGFP GFP

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


αβαβαβαβ

CD3

Wild-type RAG-1 mutant

ThymusThymus

0%T-cell

antigen

receptor

CD4

CD8

No mature TNo mature T--cellscells

Thy-1

IL-2R Precursor TPrecursor T--cellscells

No functional TNo functional T--cellscells

Immunology Lecture 1, Dr. Aguilera 2/9/2009

Technology

antigen specific antigen

j recombination dv

unique antigen antigen

bprss d

bprss23 bp

binding of antigen

mechanism of v j vd

rearranged v