Transcript Understanding the immune system A Challenge or impossible dream

Understanding the immune system
A Challenge or impossible dream
What do we (think we) understand
• Class I pathway
•
•
•
•
Proteasomal cleavage
TAP transport
Binding to MHC
Supertype clustering and epitope selection
Processing of intracellular proteins
http://www.nki.nl/nkidep/h4/neefjes/neefjes.htm
What do we NOT understand
• Class I pathway
•
•
•
•
Proteasomal cleavage
TAP transport
Binding to MHC
Supertype clustering and epitope selection
• Exceptions
•
•
•
•
K epitopes
Alternative translocation to ER
Alternative epitope splicing
Supertypes do not binding identical set of peptides
• Some alleles can not be supertype clustered
P9K ligands
• P9 of MHC ligands is generated by the
proteasome!!
• Frequency of amino acids at P9 in MHC ligands
should reflect preference for proteasomal
cleavage
•This is not the case for all amino acids
P9K ligands
P9K ligands
• P9 of MHC ligands is generated by the
proteasome!!
• Frequency of amino acids at P9 should reflect
preference for proteasomal cleavage
•This is not the case for all amino acids
• Suggests a protease other than the proteasome
is likely involved in the generation of the C-termini
of P9K ligands.
TAP independent epitope presentation
Peptides from endogenous proteins are presented
at the cell surface in complex with MHC class I
How to gain access to MHC-I
protein
-Normal entry throug TAP
-Peptides within SP gain entry
through Sec61 translocon.
MHC-I
TAP
Proteasome
-Unknown ER proteases
cleave proteins in ER
membrane or lumen
-Furin cleave proteins in
Post-ER compartment
exopeptidase
Hydrophobic
peptides
Sec6
1
-Simple diffusion across
membranes by hydrophobic
peptides
furin
-Regurgitation
-Unknown entry route
regurgitation
ER
Unknown
entry route
Datasets
From Andreas Weinzierl, Tübingen University: 40 MHC-I epitopes
eluted from the surface the human .174 cell line that doesn’t express
TAP
For comparison:
From SYFPEITHI (http://syfpeithi.bmi-heidelberg.com/): 308 MHCI epitopes eluted from the surface of normal, TAP-containing human
cells
A2
Epitope
Ex for name of
sourceprotein
(acc. to sp or nr)
Startpos
epitope
Signalpeptide
SPase
cleavage
(NN/HMM)
SPase
cleavage
site Endpos
.174/.4
5
-Normal entry throug TAP
LLSAEPVPA
CD79B_HUMAN
20
Yes
28
0
LLGPRLVLA
TMP21_HUMAN
23
Yes
31
0
ALSAYDLVL
Q6LCB5_HUMAN
29
yes
38
1
-Peptides within SP gain
253 entry through Sec61
186 translocon.
SLWGQPAEA
CO4A5_HUMAN
18
Yes
26
0
124
VLAPRVLRA
RCN1_HUMAN
21
Yes
29
0
120
ALVVQVAEA
HEXB_HUMAN
34
Yes
LLAAWTARA
A4_HUMAN
9
Yes
VLLKARLVPA
gb|AAY24258.1
19
Yes
KMDASLGNLFA
FAM3C_HUMAN
30
Yes
24
-16
LLFSHVDHVIA
NAC1_HUMAN
25
Yes
35
0
FLGPWPAAS
AMRP_HUMAN
22
Yes
28/32
-2/2
SLYALHVKA
VKOR1_HUMAN
23
Yes
26/34
-5/3
LLLSAEPVPA
CD79B_HUMAN
19
Yes
28
0
AMAPPSHLLL
gb|AAC17709.1
473
Yes
21
-461
18
FLLGPRLVLA
TMP21_HUMAN
22
Yes
31
0
18
LLLDVPTAAV
GILT_HUMAN
26
Yes
37
2
18
LLLDVPTAAVQA
GILT_HUMAN
26
Yes
37
0
15
LLDVPTAAV
GILT_HUMAN
27
Yes
37
2
14
VLFRGGPRGLLAVA
SSRA_HUMAN
19
Yes
20
-12
13
LLSAEPVPAA
CD79B_HUMAN
20
Yes
28
-1
13
AVLALVLAPAGA
NRP1_HUMAN
10
Yes
21
0
13
LAPRVLRA
RCN1_HUMAN
22
Yes
29
0
4
AALLDVRSVP
GDF5_MOUSE
269
Yes
27
-251
4
LLATLAAAML
CLP24_HUMAN
177
Yes
25
-161
0,05
28/42
17
32/28
0
0
0
557
-Unknown ER proteases
cleave proteins in ER 116
membrane or lumen
92
48
-Furin cleave proteins in
36 Post-ER compartment
28
-Simple diffusion across
23 membranes by
hydrophobic peptides
24
22
-Regurgitation
-Unknown entry route
?
Epitopes present in the N-terminal part of the SP
ER membrane
Cytosol
ER lumen
N’
C’
Sec61
ribosome
-Normal entry throug TAP
A2, cont.
Epitope
Ex for name of
sourceprotein
(acc. to sp or nr)
SignalPeptide
(SignalP)
Startos
epitope
SPase
cleavage
SPase
site cleavage Endpos
-Peptides within SP gain entry
through Sec61 translocon.
.174/.4
5
ALLSSLNDF
NIF3L_HUMAN
5
No
na
13
LLHPPPPPPPA
RANB9_HUMAN
68
No
na
13
QLQEGKNVIGL
TAGL2_HUMAN
165
No
na
8
SLPKKLALL
L10K_HUMAN
72
No
na
3
B51
Epitope
Ex for name of
sourceprotein
(acc. to sp or nr)
HGVFLPLV
K0247_HUMAN
MAPLALHLL
Startpos
epitope
Signalpeptide
(SignalP)
SPase
SPase
cleavage
cleavage site -site
Endpos
-Unknown ER proteases
cleave proteins in ER membrane or lumen
-Furin cleave proteins in
Post-ER compartment
-Simple diffusion across
membranes by hydrophobic
peptides
.174/.45
21 Yes
39
11
92
FIG1_HUMAN
1 Yes
21
12
18
MASRWGPLIG
CAB45_HUMAN
8 Yes
36
19
5
MAPRTLVL
1A02_HUMAN
4 Yes
24
13
0,5
MAPRTLIL
1C03_HUMAN
4 Yes
24
13
0,2
GSHSMRYF
1A01_HUMAN
25 Yes
24
-8
0,2
ILAPAGSLPKI
ref|XP_514384.1|
328 No
na
6
KAPVTKVAA
PDLI1_HUMAN
240 No
na
2
NPLPSKETI
TYB4_HUMAN
26 No
na
1
NPYDSVKKI
FAT10_HUMAN
25 No
na
0,2
DALDVANKIGII
RL23A_HUMAN
145 No
na
0,07
YPFKPPKV
UB2E3_HUMAN
120 No
na
0,04
-Regurgitation
-Unknown entry route
Protein with SP
Protein without SP
?
How to gain access to MHC-I
protein
-Normal entry throug TAP
-Peptides within SP gain entry
through Sec61 translocon.
MHC-I
TAP
Proteasome
-Unknown ER proteases
cleave proteins in ER
membrane or lumen
-Furin cleave proteins in
Post-ER compartment
exopeptidase
Hydrophobic
peptides
Sec6
1
-Simple diffusion across
membranes by hydrophobic
peptides
furin
-Regurgitation
-Unknown entry route
regurgitation
ER
Unknown
entry route
Presentation of alternatively spliced
epitopes
Presentation of Noncontiguous
peptides
• The conventional approach to epitope
discovery is to use overlapping peptides
• What if splicing of noncontiguous peptides
occure?
HLA-A3 Antigen produced by
splicing of Noncontiguous peptides
Warren et al. Science, 313, p 1444, 2006
HLA-A3 Antigen produced by
splicing of Noncontiguous peptides
NetMHC version 3.0. Prediction using Neural Networks. Allele A0301.
Strong binder threshold 50.00. Weak binder threshold 500.00.
-------------------------------------------------pos
peptide 1-log50k(aff) affinity(nM) Bind Level
-------------------------------------------------0 STPKRRHKK
0.4237
510
1 TPKRRHKKK
0.1019
16598
2 PKRRHKKKS
0.0071
46309
3 KRRHKKKSL
0.0082
45761
4 RRHKKKSLP
0.0137
43097
5 RHKKKSLPR
0.1051
16035
6 HKKKSLPRG
0.0085
45624
7 KKKSLPRGT
0.0091
45326
8 KKSLPRGTA
0.0109
44425
9 KSLPRGTAS
0.0991
17110
10 SLPRGTASS
0.0608
25887
11 LPRGTASSR
0.0732
22656
--------------------------------------------------
Identity
A3
A3
A3
A3
A3
A3
A3
A3
A3
A3
A3
A3
Number of high binders 0. Number of weak binders 0. Number of peptides 12
Warren et al. Science, 313, p 1444, 2006
Antigen produced by splicing of
Noncontiguous peptides
Warren et al. Science, 313, p 1444, 2006
Antigen produced by splicing of
Noncontiguous peptides
Final peptide: SLPRGTSTPK
A3 motif: P2:L, P9:K
Warren et al. Science, 313, p 1444, 2006
HLA-A3 Antigen produced by
splicing of Noncontiguous peptides
NetMHC version 3.0. Prediction using Neural Networks. Allele A0301.
Strong binder threshold 50.00. Weak binder threshold 500.00.
-------------------------------------------------pos
peptide 1-log50k(aff) affinity(nM) Bind Level
-------------------------------------------------0 SLPRGSTPK
0.5029
216
WB
--------------------------------------------------
Identity
SLPRGSTPK
Number of high binders 0. Number of weak binders 1. Number of peptides 1
Warren et al. Science, 313, p 1444, 2006
Supertypes. What are they good
for?
• Alleles within supertypes present the same
set of peptides!
Clustering of HLA alleles
O Lund et al., Immunogenetics.
Supertypes. What are they good
for?
• Alleles with in supertypes present the same
set of peptides!
• Is this really so?
• Less that 50% of A6802 binders will bind to
A0201!
• Less than 33% of A0201 binders will bind to
A6802!
The truth about supertypes!
A3
A26
A24
A2
A1
Supertypes are good for getting
funding, but..
• Need to define more refined method for
identifying promiscuous epitopes
• Need to develop method to predict binding
across all HLA alleles
• Supertypes is too simple a picture
What more do we (think we)
understand
• Why are epitopes 9 amino acids long?
• Why did nature not choose 15mers?
• Which class I presented peptides can bind TCR?
• Or can we estimate TCR cross reactivity?
Why 9mers?
• Why did the immune system settle on
presentation of 9mer peptides?
• The proteasome generates mostly fragments of
4-7 amino acids
• TAP preference peptides of 8-18 amino acids
• MHC preference peptides of 8-12 amino acids
• So why 9?
Information processing in the
immune system
• How many different self peptides do we
have?
• How much information is present in a 9mer?
• Can you discriminate self from non-self
based on the information in 9-mers?
Burroughs, De Boer & Kesmir, Immunogenetics, 2004, 56(5):311-20
Size of self
107 << 209 : self is a small fraction of peptide space:
Overlap with other “selfs” expected to be small.
Size of self - continued
Discriminating self
9-mers provide enough information to discriminate:
Average overlap human and pathogens <1%
(Overlap between unrelated bacteria also 1%)
Overlap with human depend on
evolutionary distance
Conclusions
• Information in 9-mers sufficient to
discriminate self from non-self
• The chance that an immunodominant
pathogenic peptide resembles self < 0.5%
TCR self-tolerance
• What determines if a MHC ligand can elicit
an immune responds?
• Similarity to self peptide
• What is similar?
• What amino acids determine similarity?
• How broad is the cross reactivity?
xx
TCR recognition of MHC:peptide
complex
TCR data processing
Experimental data
Normalization &
frequency conversion
or
Visualization (logo)
G10,
INFg, APL
G10
Cr, APL
161
cr, PSCL
T5
INFg, APL
2G4
Cr, APL
T5
CR, APL
B7
Cr, APL
PBMC
INFg, APL
3F4
Cr, PSCL,
Amididated C-terminus
Shannon information
5
Shannon Information
4.5
4
3.5
3
data
2.5
median
2
1.5
1
0.5
0
0
1
2
3
4
5
6
Peptide position
7
8
9
10
TCR and MHC specificity profile
anti-correlates
HLA-A0201 restricted
HIV Gag CTL clone
HLA-A0201
Predict TRC cross reactivity
•
TCR cross-reactivity can
(partly) be characterized from
a specificity profile, and amino
acid conservation.
• Use cross reactivity model to
predict cross reactivity
• Explain lacking
immunogenecity of predicted
CTL epitopes
• Overlap between cross
reactivity space and host
genome
Conclusions
• We might thinks we understand parts of
the immune system, but nothing is ever
always as we would like