Innovative methods to identify specific epitopes and associated antigens from a variety of different disease applications and strategies to apply this technological framework to the study of alopecia areata.

1. 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

2. Large scale epitope identification
suggests new vaccine targets
2
Lindestam Arlehamn et al. PLoS Pathog. 2013

3. Large scale epitope identification
enables novel insights in T cell biology
IKZF2!ADAM12!
40!
40!
40!
40!
40!
0!
4!
4!
4!
4!
4!
0!
RORC!
500!
0!
250!
0!
125!
250!
IL17RE!
40!
40!
40!
40!
40!
0!
200!
0!
400!
0! 0!
Th2!
Th*!
Th17!
Th1!
Tet+ !
IL12RB2!CCR2! IL23R!
Th2!
Th*!
Th17!
Th1!
120!
Tet+ !
120!
120!
120!
120!
0!
40!
40!
40!
40!
40!
0!
4!
4!
4!
4!
4!
0!
120!
60!
0"
400!
200!
0"
400!
200!
0"
BAFF(TNFSF13B)!KIT(CD117)! TIGIT!
120!
120!
120!
120!
120!
0!
40!
40!
40!
40!
40!
0!
4!
4!
4!
4!
4!
0!
150!
75!
0"
300!
150!
0"
500!
250!
0"
0! 0! 0!
mRNAnormalizedcounts!
0!
ZBTB7B (ThPOK)!
70!
70!
70!
70!
70!
0!
0!
200!
400!
15!
15!
15!
15!
0!
0!
400!
800!
15!
50!
0"
100!
280!
320!
//!
//!
ABCB1(MDR1)!
15!
15!
15!
15!
15!
0!
300!
0!
150!
B
IKZF2!ADAM12!
40!
40!
40!
40!
40!
0!
4!
4!
4!
4!
4!
0!
RORC!
500!
0!
250!
0!
125!
250!
IL17RE!
40!
40!
40!
40!
40!
0!
200!
0!
400!
EOMES!TBX21!
Th2!
Th*!
Th17!
Th1!
120!
Tet+ !
120!
120!
120!
120!
0!
40!
40!
40!
40!
40!
0!
600!
300!
0!
300!
150!
0!
Th2!
Th*!
Th17!
Th1!
Tet+ !
IL12RB2!CCR2! IL23R!
Th2!
Th*!
Th17!
Th1!
120!
Tet+ !
120!
120!
120!
120!
0!
40!
40!
40!
40!
40!
0!
4!
4!
4!
4!
4!
0!
120!
60!
0"
400!
200!
0"
400!
200!
0"
BAFF(TNFSF13B)!KIT(CD117)! TIGIT!
120!
120!
120!
120!
120!
0!
40!
40!
40!
40!
40!
0!
4!
4!
4!
4!
4!
0!
150!
75!
0"
300!
150!
0"
500!
250!
0"
GZMK!GZMA! GZMM!
120!
120!
120!
120!
120!
0!
40!
40!
40!
40!
40!
0!
4!
4!
4!
4!
4!
0!
4000!
2000!
0!
3000!
1500!
0!
80!
40!
0!
PRF1, GZMK, GZMA, EOMES,TBX21!GZMM!RORC, ADAM12, IL17RE!
mRNAnormalizedcounts!
PRF1!
120!
120!
120!
120!
120!
0!
2000!
0!
1000!
ZBTB7B (ThPOK)!
70!
70!
70!
70!
70!
0!
0!
200!
400!
15!
15!
15!
15!
0!
0!
400!
800!
15!
50!
0"
100!
280!
320!
//!
//!
IKZF2!CCR2, IL12RB2, IL23R, KIT, BAFF,ABCB1!TIGIT, ZBTB7B!Examples: !
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

4. 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
4
Weiskopf et al. PNAS 2013
Magnitude per responder
0 5 10 15 20
0
1000
2000
3000
4000
5000
p= 0.04
Protection ------- Susceptibility
AverageSFC/responder
Frequency of responses
20
30
40
p= 0.36
ofresponders[%]
Magnitude per Epitope
0 5 10 15 20
0
100
200
300
400
500
p= 0.02
Protection ------- Susceptibility
AverageSFC/epitope
Breadth of response
15
20
25
p= 0.2
numberof
/responder

5. 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

6. 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

7. A majority of Th2 cells in allergic
donors target novel epitopes
7
IL-5
TGExtract
1
2
3
4
5
1
2
3
4
5
6
7
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42
0
20
40
60
80
100
%Responders
Allergics
Normals
Known
allergens
Novel
antigens
IL-5
Allergics Normals
0
500
1000
1500
2000
2500
SFC/106PBMC
Known
Novel
A
B
Novel antigens are therapeutic in allergy
models

8. 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

9. 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…

10. 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

11. 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

12. 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

13. 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

14. Peptide selection strategy
311 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.org
MHC binding tool
v. 2.15.1

15. 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

16. Acknowledgments
• Sinu Paul
• John Sidney
• April Frazier
• Cecilia Lindestam Arlehamn
• AA donors
• LJI clinical coordination team
• Angela Christiano
• Annemieke De Jong

18. 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