large scale epitope identification screen and its potential application to the study of alopecia...
TRANSCRIPT
Technology is available to generate targeted experimental data
• Technology advancement allow high throughput antigen and epitope identification
• Combination of bioinformatics, proteomics, next generation sequencing and high throughput assays
• Several NIAD contracts tackled biodefense targets, emerging and reemerging diseases, and allergens
• Field moving past anecdotal evidence, into population based studies
Large scale epitope identification suggests new vaccine targets
2Lindestam Arlehamn et al. PLoS Pathog. 2013
Large scale epitope identification enables novel insights in T cell biology
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Figure 4
IKZF2!ADAM12!
40!
40!
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40!
0!
4!
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RORC!
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IL17RE!
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Th* > All!Th* < All!
Th1 vs Th17 gene signature !
Th
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Th
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!T
h2
!T
h*&
Te
t+
!
Th1! Th17!
Th*gene signature!
Su
bje
cts
!
HC !
LTBI!
Th* ˜ Th1! Th*! ˜ Th17!
EOMES!TBX21!
Th2!
Th*!
Th17!
Th1!
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Tet+ !
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Th2!
Th*!
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Tet+ !
IL12RB2!CCR2! IL23R!
Th2!
Th*!
Th17!
Th1!
120!
Tet+ !
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0!
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BAFF(TNFSF13B)!KIT(CD117)! TIGIT!
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GZMK!GZMA! GZMM!
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PRF1, GZMK, GZMA, EOMES,TBX21!GZMM!RORC, ADAM12, IL17RE!
mR
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lize
d c
oun
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PRF1!
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ZBTB7B (ThPOK)!
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IKZF2!CCR2, IL12RB2, IL23R, KIT, BAFF,ABCB1!TIGIT, ZBTB7B!Examples: !
TT
T!
T!
T! Tet+!
ABCB1(MDR1)!
15!
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Figure 4
IKZF2!ADAM12!
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RORC!
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IL17RE!
40!
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Log
2 F
C!
Th* > All!Th* < All!
Th1 vs Th17 gene signature !
Th
1!
Th
17
!T
h2
!T
h*&
Te
t+
!
Th1! Th17!
Th*gene signature!
Su
bje
cts
!
HC !
LTBI!
Th* ˜ Th1! Th*! ˜ Th17!
EOMES!TBX21!
Th2!
Th*!
Th17!
Th1!
120!
Tet+ !
120!
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120!
0!
40!
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Th2!
Th*!
Th17!
Th1!
Tet+ !
IL12RB2!CCR2! IL23R!
Th2!
Th*!
Th17!
Th1!
120!
Tet+ !
120!
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0!
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BAFF(TNFSF13B)!KIT(CD117)! TIGIT!
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GZMK!GZMA! GZMM!
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PRF1, GZMK, GZMA, EOMES,TBX21!GZMM!RORC, ADAM12, IL17RE!
mR
NA
no
rma
lize
d c
oun
ts!
PRF1!
120!
120!
120!
120!
120!
0!
2000!
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ZBTB7B (ThPOK)!
70!
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0"
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//!
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IKZF2!CCR2, IL12RB2, IL23R, KIT, BAFF,ABCB1!TIGIT, ZBTB7B!Examples: !
TT
T!
T!
T! Tet+!
ABCB1(MDR1)!
15!
15!
15!
15!
15!
0!
300!
0!
150!
Increased cell survival and proliferationIncreased resistance to TB
Increased persistence
Cytotoxic CD4+
Lindestam Arlehamn et al. JI 2014
Large scale epitope identification enables definition of correlates of protection
• High resolution map of T cell responses in the general population of an endemic area (Sri Lanka)
• 408 epitopes described, 80% novel
• Over 700 patients from hyperendemic areas
• CD8 T cells are associated with protection from DENV
4Weiskopf et al. PNAS 2013
0 5 10 15 200
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5001000
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Average response per HLA
p= 0.05
Protection ------- Susceptibility
Av
era
ge
SF
C/ H
LA
Magnitude per responder
0 5 10 15 200
1000
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5000p= 0.04
Protection ------- Susceptibility
Av
era
ge
SF
C / r
es
po
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Frequency of responses
0 5 10 15 200
10
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40p= 0.36
Protection ------- Susceptibility
Fre
qu
en
cy
of
res
po
nd
ers
[%
]
Magnitude per Epitope
0 5 10 15 200
100
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500p= 0.02
Protection ------- Susceptibility
Avera
ge S
FC
/ ep
ito
pe
Breadth of response
0 5 10 15 200
5
10
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25
p= 0.2
Protection ------- Susceptibility
Av
era
ge
nu
mb
er
of
ep
ito
pe
s / r
es
po
nd
er
Novel protein identification immunoproteomics
• Pollen extract separated on 2D gel
• Spots picked, based on antibody or protein staining
• Spots cut out from gel, analyzed in mass spectrometer
• 83 new proteins from 2D gel + 10 proteins from whole extract mass spec were chosen for further studies
Schulten et al. PNAS 2013
T cell antigen identification based on HLA class II binding predictions
• Predict peptides binding to a panel of 25 HLA class II molecules (DR, DP, DQ)
• Synthesize 822 peptides that bind promiscuously (>12 HLA variants)
• Test peptides as pools for IL-5 production in PBMC from TG allergic donors
A majority of Th2 cells in allergic donors target novel epitopes
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IL-5
TG
Ext
ract 1 2 3 4 5 1 2 3 4 5 6 7 8 9
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% R
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on
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rs
Allergics
Normals
Known allergens
Novel antigens
IL-5
Allergics Normals0
500
1000
1500
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2500
SF
C/1
0 6
PB
MC
Known
Novel
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Novel antigens are therapeutic in allergy models
Towards large-scale screen of potential targets for T cell recognition in AA
• The issue of large versus small
• Donor recruitment
• HLA typing of donor cohort
The issue of large versus small for large-scale screens
• According to one approach, it is most relevant to study in situ T cells during acute episodes
• High in biological relevance - but not suited to high throughput epitope/antigen identification
• Memory and resident T cells, especially away from acute phases, recirculate in the periphery and they are readily detected in PBMC
• Examples from TB, allergies, influenza, herpes…
Donor recruitment• Based on these considerations we moved to
enroll AA donors through community outreach
• The AA community is in general eager to help, amenable to full unit donations
Reported HLA associations in AA
• Increased frequency of DQB1*03, coding for DQ7
heterodimers in patients when compared with
controls British Journal of Dermatology 165(4):823-7
• HLA-DR4, DR11 and DQ*03 alleles increased in
unrelated AA patients compared with controls. Journal of
Inv. Derm. Symp.Proc. Vol 4;3, December 1999
• Most recent metanalysis Betz RC et al. Nat Commun. 2015
Jan 22;6:5966
• Class II association, but CD8 infiltrate -> a conservative
approach would target both
HLA typing of donor cohort
Allele Genes GF alopecia GF common
A*01:01 1 2.8 8.5
A*02:01 13 36.1 13.5
A*02:05 2 5.6 1.2
A*02:06 1 2.8 2.5
A*03:01 1 2.8 8.0
A*11:01 6 16.7 6.7
A*24:02 4 11.1 8.8
A*24:159 1 2.8
A*25:01 1 2.8 1.2
A*29:02 2 5.6 1.5
A*30:01 2 5.6 2.6
A*32:01 1 2.8 2.9
A*68:01 1 2.8 2.3
Expected
3
5
0
1
3
2
3
0
0
1
1
1
1
(p=0.02) not bonferroni corrected
A list of over 300 potential targets
• Compiled from published proteomic studies, gene expression data (genes down in AA vs. control scalp), and several additional hair follicle proteins
• Many keratins and keratin-associated proteins– Because of protein homology a more limited set of
peptides maybe required
• Trichohyalin and keratins are heavily modified– We do not know which proteins are modified and where
/how
– we focused on unmodified versions, hoping to detect reactivity against non-modified peptides
Peptide selection strategy311 unique protein sequences (UniProt) –
Clustered at 50% identity threshold (UCLUST)
15-mers overlapping by 10aa + variants from alignment
Predictions for general Class II DR & A*02:01
2278 MHC class II peptides
(10%-ile +DQB1*03:01)
2000 MHC class I
(1%-ile)
www.iedb.orgMHC binding tool v. 2.15.1
Overall message
• Technology is available to generate targeted experimental data
• We have recruited an initial donor cohort (and age matched controls)
• Assembled a target set of over 300 proteins
• The IEDB analysis resource can be used to predict epitopes
Acknowledgments
• Sinu Paul
• John Sidney
• April Frazier
• Cecilia Lindestam Arlehamn
• AA donors
• LJI clinical coordination team
• Angela Christiano
• Annemieke De Jong
HLA typing of donor cohort
Allele Genes GF alopecia GF SD GF common Expected
DRB1*01:01 2 5.6 4.7 2.8 2
DRB1*01:03 1 2.8 0.4 0.4 0
DRB1*03:01 1 2.8 8.0 7.1 3
DRB1*04:01 2 5.6 5.3 2.3 2
DRB1*04:07 2 5.6 1.8 2.4 1
DRB1*07:01 5 13.9 8.0 7.0 3
DRB1*08:03 1 2.8 0.6 3.8 0
DRB1*08:04 1 2.8 0.7 1.1 0
DRB1*10:01 1 2.8 1.1 1.9 0
DRB1*11:01 3 8.3 5.6 6.1 2
DRB1*11:02 1 2.8 0.7 1.1 0
DRB1*11:04 2 5.6 2.3 1.4 1
DRB1*12:01 1 2.8 2.2 2.0 1
DRB1*13:01 2 5.6 5.5 3.2 2
DRB1*13:15 1 2.8 0.0 0
DRB1*14:01 1 2.8 3.4 0
DRB1*14:02 1 2.8 2.8 0
DRB1*14:06 1 2.8 0.2 0.6 0
DRB1*15:01 6 16.7 14.2 6.3 5
DRB1*16:02 1 2.8 0.4 3.9 0