immunology lecture 1, dr. aguilera 2/9/2009
TRANSCRIPT
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
{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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
�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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
�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)
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Fig. 2 Schatz and Baltimore (1988)
TRX-1TR-1
+ -
gpt Ja JbVκ
gpt
RSSs
VκJa
Fig. 3 Schatz and Baltimore (1988)
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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
Immunology Lecture 1, Dr. Aguilera 2/9/2009
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