Transcript Christina Sylvester

“Theoretical and
Experimental
“Experimental
and
Theoretical
description of
Peptide-MHC binding”
Christina Sylvester-Hvid, IMMI, Panum
Generation of Recombinant MHC
Class I and Characterization of the
People
Interaction with Peptide
- Contributions to the Human MHC project
Christina Sylvester-Hvid, IMMI, Panum
MHC class I with peptide
Peptide
Binding
groove
Christina Sylvester-Hvid, IMMI, Panum
Peptides are bound to MHC class I
molecules by their ends
N
C
Christina Sylvester-Hvid, IMMI, Panum
Peptides are bound to MHC class II molecules by
interactions along the length of the binding groove
N
C
Christina Sylvester-Hvid, IMMI, Panum
Peptide binding grove
TcR
Hydrogen bonds
Salt bridge
Hydrogen bonds
Pockets
Lauemøller, S.L. and Buus, S. 2001
Christina Sylvester-Hvid, IMMI, Panum
Peptides bind to MHC molecules through
structurally related anchor residues
Two MHC
class I alleles
(P2 (Y), (Pc): V,I, L)
Christina Sylvester-Hvid, IMMI, Panum
Peptides that bind to MHC-II molecules are
variable in length
Christina Sylvester-Hvid, IMMI, Panum
Comparison of the cleft architectures of
murine class I alleles, Kb and Kk
Stryhn A, et al., 1996, PNAS
Peptide: RGYVYQGL
Peptide: FESTGNLI
The peptide is deeply embedded
Christina Sylvester-Hvid, IMMI, Panum
Most of the peptide is hidden in the groove - only a
minor part is available for the TcR.
FES T GN L I
Available for the TCR
Top view Mouse class I Kk
in complex with peptide FESTGNLI
Christina Sylvester-Hvid, IMMI, Panum
Peptide binding grove
Peptides can be anchor optimized, affinity can increased X 10,
Does not changes the T cell specificity!
Hydrogen bonds
Salt bridge
Hydrogen bonds
Pockets
GK
Lauemøller, S.L. and Buus, S. 2001
Christina Sylvester-Hvid, IMMI, Panum
Peptides - prime targets of immune recognition
TcR
Peptide
HLA
Christina Sylvester-Hvid, IMMI, Panum
Determining primary protein structures
is like
charting the landscape of the immune system
Christina Sylvester-Hvid, IMMI, Panum
From proteins to immunogens
1/200
1/200 peptides ends up in the MHC binding groove
Christina Sylvester-Hvid, IMMI, Panum
Translating genomes to immunogens
Christina Sylvester-Hvid, IMMI, Panum
HLA polymorphism - alleles
A total of
245 HLA-A
480 HLA-B
117 HLA-C
class I alleles have been assigned.
A total of
3 HLA-DRA, 380 HLA-DRB
22 HLA-DQA1, 52 HLA-DQB1
20 HLA-DPA1, 97 HLA-DPB1
class II alleles have been assigned.
As of April 2002 (http://www.anthonynolan.com/HIG/index.html)
Christina Sylvester-Hvid, IMMI, Panum
HLA polymorphism - supertype specificity
Supertype
Specificity
Av. frequency
P2
Pc
A1
TI (LVMS)
FWY
25 %
A2
AILMVT
AILMVT
42 %
A3
AILMVST
RK
44 %
A24
YF (WIVLMT)
FI (YWLM)
40 %
B7
P
AILMVFWY
50 %
B27
RHK
FYL (WMI)
23 %
B44
E (D)
FWYLIMVA
37 %
B58
ATS
FWY (LIV)
10 %
B62
QL (IVMP)
FWY (MIV)
18 %
Sette et al, Immunogenetics (1999) 50:201-212
Christina Sylvester-Hvid, IMMI, Panum
Bindings affinity vs. number of epitopes
TB: ~ 4000 proteins ~ 1.33 mill. aa ~ 4 mill. 8-10´mers
HIV ~ 9 proteins ~ 3000 aa ~ 9000 8-10´mers
Number of
peptides
KD =
< 50 nM
< 250 nM
< 500 nM
< 5000 nM
< 50000 nM
Increasing peptide affinity
Christina Sylvester-Hvid, IMMI, Panum
Experimental description of
peptide-MHC binding
Many different peptide-MHC-binding assays have been suggested over the
years ; without being exhaustive:
1) Olsen, A.C., et al., Eur. J. Immunol. 1994; 24:385-392
2) Buus, S et al., J. Immunol. 1982; 129:1883-1891.
3 ) Buus, S. and Werdelin, O. J. Immunol. 1986; 136: 459-465.
4) Babbitt, B.P et al., Proc. Natl. Acad. Sci. USA. 1986; 83:4509-4513.
5) Buus, S., et al., Cell. 1986; 47:1071-1077.
6) Luescher, I. F et al., Proc. Natl. Acad. Sci USA. 1988; 85:871-874.
7) Townsend, A., et al., Nature. 1989; 340:443-448.
8) Bouillot, M. et al.,Nature. 1989; 339:473-475.
9) Busch, R. and Rothbard, J. B. J. Immunol. Meth. 1990: 134:1-22.
10) Townsend, A., et al., Cell 1990:62:285-295.
11) Parker, K.C. et al., J. Immunol. 1992; 49:1896-1904.
12) Joosten, I. Et al., Trans. Proc. 1993; 25:2842-2843.
13) Khilko, S.N. et al., J. Biol. Chem. 1993; 268:15425-15434.
14) Regner, M. et al.,Exp Clin Immunogenet. 1996; 13:30-35.
Christina Sylvester-Hvid, IMMI, Panum
RMAS Assay: classical way to measure peptide binding
- However not quantitative (no determination of the affinity)
At 37 °C
At 26 °C
Add peptide
TAP difficient cell line
Measure T cell activation
Christina Sylvester-Hvid, IMMI, Panum
Experimental description of
peptide-MHC binding
How to examine HLA specificity?
”What the HLA has bound in vivo”
 Elution and sequencing of natural ligands
 Simpel motif ~ low sensitivity predictions
Hans-Georg
Rammensee et al.,
www.syfpeithi.de
”What the HLA will, or will not, bind in vitro”
 Determine the binding strength of any peptide
 Extended motif ~ higher sensitivity predictions
Søren Buus et al.,
www.cbs.dtu.dk/services/NetMHC/
Christina Sylvester-Hvid, IMMI, Panum
”What the HLA has bound in vivo”
Prediction of binding, web based services
(non quantitative)
Christina Sylvester-Hvid, IMMI, Panum
www.syfpeithi.de (Hans-Georg Rammensee et al.,)
Christina Sylvester-Hvid, IMMI, Panum
Søren Buus et al.,
www.cbs.dtu.dk/services/NetMHC/
Scanning the genome of Chlamydia pneumonia for CTL epitopes
Acc #
gi|8163394
gi|7189942
gi|7189608
gi|8163474
gi|7189405
gi|8163540
gi|7189969
gi|7189995
gi|8163481
gi|7189705
gi|7189594
gi|7189985
gi|7189979
gi|7189388
gi|7190008
gi|7189561
gi|7189544
gi|7189647
gi|8163547
gi|7189892
Protein
position
850
120
96
40
496
101
360
149
31
238
407
159
269
364
203
318
52
296
56
Kk binding
sequence
7 EEHGSTTI
EELDASAI
EEVYMGTI
TELLAEYI
AEQLASEI
EEVFSGFI
EELWAAEI
EEAKSAFI
EELRAVSI
EEIRYRII
DERYASWI
EELGSEAI
LERLAGFI
FELVAHIG
EERLAIFI
WEVVSHFI
DEVIARIH
FEVLCRDI
EEFRQGYI
EELFADFI
(nM)
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Proteasome
C-flush
Survival
0.73
0.94
0.89
0.26
0.28
0.29
0.19
0.35
0.74
0.08
0.03
0.88
0.16
0.16
0.04
0.60
0.13
0.10
0.01
0.30
0.01
0.45
0.03
0.10
0.01
0.32
0.03
0.08
0.00
0.89
0.00
0.43
0.00
0.15
0.00
0.02
0.00
0.38
0.00
0.53
Efficacy
3
9
26
33
44
72
75
107
161
537
591
604
720
740
789
962
1662
2023
2100
2300
How to determine peptide affinity
Law of mass action
koff
[R] + [L]
[RL]
kon
koff
[MHC] + [P]
[P*MHC]
kon
KD = koff (S-1)/kon (M-1S-1)
100%
Saturation
assay
Inhibition
assay
50%
Peptide [M]
KD = (10-15-10-6 M)
Peptide Log [M]
Cold Peptide Log [M]
Log IC50
Christina Sylvester-Hvid, IMMI, Panum
How to do radioactive biochemical
inhibition binding assays
• Obtain purified HLA
• Or recombinant heavy chain & b2m
• Obtain indicator peptide
• Perform dose titration of any inhibitory peptide
• Separate free from bound peptide
• Calculate binding and IC50
Binding test Peptide
Non binding test
peptide
Christina Sylvester-Hvid, IMMI, Panum
A spun column binding assay
MHC
b2m
peptide
G50
Non binding
test peptid
Binding test
peptid
Christina Sylvester-Hvid, IMMI, Panum
How to determine the peptide binding motif
Christina Sylvester-Hvid, IMMI, Panum
Specificity description of A*0204
(matrix)
Christina Sylvester-Hvid, IMMI, Panum
The radioactive biochemical binding assay
PROS
CONS
• Truly quantitative
• Radioactive
• Can address affinities in
the low nM level
• Not a standard method
• Waste problem
• Reproducible
Christina Sylvester-Hvid, IMMI, Panum
The Quantitative ELISA Capable of
Determining Peptide-MHC Class I Interaction
• Made possible by our recent development of highly
active recombinant MHC class I heavy chains
– functional equivalents of ”empty” molecules
L.O.Pedersen et al., , EJI. 2001, 31: 2986
• Pros:
• Reasonably simple, sensitive and
quantitative
• Does not depend on labeled peptide
• It is easily adaptable to other laboratories
• Disseminated protocol and standard reagents
Christina Sylvester-Hvid, IMMI, Panum
Strategy for the assay
• Step I: Folding of MHC class I molecules in solution
Incubation
• Step II: Detection of de novo folded MHC class I molecules by ELISA
Development
Sensitivity below 0.1 nM or 5 x 10-15 M
MHC class I complex
! Sylvester-Hvid, IMMI, Panum
Christina
Concentrations of de novo folded MHC complexes, plotted as
function of the concentrations of peptide offered
nM MHC complex detected
Fitted in Prism® 4.0 GraphPad
Data out put:
BMAX : Amount of detected complex
including 95% confidence interval
KD: Peptide affinity
including 95% confidence interval
R2: Precision of the fit
nM peptide offered
C.Sylvester-Hvid, et al., Tissue Antigens 2002. 59: 259
Christina Sylvester-Hvid, IMMI, Panum
Data base of HLA ligands, founded by
the NIH (Nat. Inst. Health) USA
Christina Sylvester-Hvid, IMMI, Panum
Take home messages….
 MHC class I molecule preferably binds peptides of 8-11 aa
 Peptides bind to the MHC binding groove by hydrogen
bonds, hydrophobic forces and other non-covalent
interactions
 MHC binding specificity is obtained through the
recognition of peptide- motifs, a recognition mode which
requires the presence and proper spacing of particular
amino acids in certain anchor positions.
 The binding strength, the affinity - is very important: The
higher affinity of a peptide to the class I molecule, the higher
chance of being immunogenic.
Christina Sylvester-Hvid, IMMI, Panum
and more…..
http://www.ihwg.org/components/peptider.htm
 Peptide binding can be addressed in a qualitative or
quantitative matter.
 The Radioactive binding assay or The quantitative ELISA
assay can measure the exact affinity in nM for the best known
binders
 Measurements of affinity can be used to generate tools
(ANN) for prediction of peptide binding to any MHC class I
molecule of human interest.
 Subsequently to validation, peptides can be directly used
in peptide based vaccines or rationally optimized to increase
their immunogenesity
Christina Sylvester-Hvid, IMMI, Panum