www.sciencetranslationalmedicine.org/cgi/content/full/5/216/216ra176/DC1

Supplementary Materials for

CD4+ T Cell Autoimmunity to Hypocretin/Orexin and Cross-Reactivity to a 2009 H1N1 Influenza A Epitope in Narcolepsy

Alberto K. De la Herrán-Arita, Birgitte Rahbek Kornum, Josh Mahlios, Wei Jiang, Ling Lin, Tieying Hou, Claudia Macaubas, Mali Einen, Gi seppe Plazzi, Catherine Crowe,

Evan W. Newell, Mark M. Davis, Elizabeth D. Mellins,* Emmanuel Mignot*

*Corresponding author. E-mail: mignot@stanford.edu (E.M.); mellins@stanford.edu (E.D.M.)

Published 18 December 2013, Sci. Transl. Med. 5, 216ra176 (2013) DOI: 10.1126/scitranslmed.3007762

The PDF file includes:

Fig. S1. CD4+ T cell TNF-α response to DQ0602 strong binding peptides. Fig. S2. T cell response to HCRT epitopes with different APCs. Fig. S3. ROC curves for ELISpot data. Fig. S4. DQ0602 binding of HCRT and pHA1 peptides with amino acid substitutions. Fig. S5. Binding of pH1N1 peptides to DQ0602. Fig. S6. Possible mimics of HCRT epitopes in pH1N1 stimulate T cells from narcoleptic patients and controls. Fig. S7. Representative ELISpot images corresponding to Figs. 4 and 6. Table S1. Prepro-HCRT peptides showing binding to DQ0602. Table S2. pHA1 peptides showing binding to DQ0602. Table S3. pNA1 peptides showing binding to DQ0602. Table S4. pPB1 peptides showing binding to DQ0602. Table S5. Influenza A H1N1 strains used for vaccinations by year. Reference (48)

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1

Supplementary Materials

Figure S1. CD4+ T cell TNF-α response to DQ0602 strong binding peptides.

CD4+ responses to HCRT56-68, HCRT87-99, and EBV490-503 when presented by T2.DQ0602

cells were tested in 4 narcoleptic patients and 2 healthy controls. Right panel displays

representative ELISpot images. Shown is number of Spot Forming Units (SFU) per 105 T

cells.

2

Figure S2. T cell response to HCRT epitopes with different APCs.

(A) IFN-γ ELISpot data from stimulations of 7 patient samples vs. 7 control samples with

HCRT56-68 and HCRT87-99 peptides added to either total PBMCs (containing both CD4+ T

3

cells, CD8+ T cells and autologous antigen presenting cells), autologous dendritic cells

(DCs) with purified CD4+ T cells, or T2.DQ0602 cells with purified CD4

+ T cells.

Shown is number of Spot Forming Units (SFU) per 105 T cells. Mann-Whitney U test:

*P

4

Figure S3. ROC curves for ELISpot data.

Using receiving operating characteristics curve analysis, a statistical method to optimize

sensitivity and specificity, we found that a cut off for HCRT56-68 ≥ 5 (A) and HCRT87-99 ≥1

5

(B) Spot Forming Units (SFU) per 105 CD4

+ T cells, had sensitivities of 0.83 [0.67-1.0] and

0.92 [0.78-1.0] and specificities of 0.96 [0.88-1.0] and 0.88 [0.72-1.0], respectively.

Combining both readings, HCRT56-68 ≥ 3 and HCRT87-99 ≥ 2 (C) SFU/105

cells had a

sensitivity of 0.83 [0.63-0.96] (all but four patients positive) and a specificity of 1.0 [1.0-

1.0] (none of the controls met criteria); while HCRT56-68 ≥ 5 or HCRT87-99 ≥ 1 (D)

SFU/105cells had a sensitivity of 0.96 [0.88-1.0] and a specificity of 0.84 [0.70-0.96].

6

7

Figure S4. DQ0602 binding of HCRT and pHA1 peptides with amino acid substitutions.

(A) Peptides with amino acid substitutions at each residue position (1-9) of HCRT56-68

were tested for their ability to bind DQ0602, as determined by EBV490-503 competition.

(B-C) Peptides with phenylalanine substitutions at each residue position of (B) HCRT87-99

and (C) pHA1275-287 were tested for their ability to out-compete EBV490-503 binding to

DQ0602 (47). (D) Schematic overview of the substitutions tested in HCRT56-68, HCRT87-99,

and pHA1275-287 and their effect on DQ0602 binding. All experiments where performed

2-3 times in technical duplicates or triplicates. *= peptides difficult to dissolve in assay

buffer.

8

Figure S5. Binding of pH1N1 peptides to DQ0602.

(A-C) Binding of peptides from pH1N1 proteins to DQ0602. Overlapping 15-mer peptides

from the pHA1 (A), pNA1 (B), and pPB1 (C) proteins in A/California/7/2009 (pH1N1)

were tested for competition with EBV490-503 for DQ0602 binding in vitro. Binding was

expressed as the percentage of the reference peptide (EBV490-503) displaced; >75%

decrease in signal is good binding, 50-75% moderate binding, and

9

Figure S6. Possible mimics of HCRT epitopes in pH1N1 stimulate T cells from

narcoleptic patients and controls.

(A) Representative examples of IFN-γ ELISpot results of other DQ0602 binders from

pH1N1. Data are obtained using NA13-11, NA181-90, HA174-83, and PB111-20, from 5 patients

and 3 controls. For details see methods.

SFU

/10

CD

4 T

-cel

ls

FIGURE S6

pHA1275-287

Peptide concentration (nM)1x10

21x10

31x10

11x10

01x10

-11x10

-21x10

-31x10

-4

0

1

3

7

15

31

63

127

255

C. Peptide dose-response of CD4 T-cells

ELISpot representative examples

PATIENTCONTROL

2009 20112012 20122000

NA13-11

NA181-90

HA174-83

PB111-20

-

47 3095

31 5101

20 1815

53 2332

HCRT56-68HCRT87-99

Blood Sample YearDisease Onset Year

SFU

/10

CD

4 T

-cel

ls5

+

0

1

3

7

15

31

63

127

255

511

pHA1275-283

Control

Patient

PMA &Ionomycin

No peptide

n= 35 n= 37 n= 35 n= 37

B. CD4+ T-cell IFN-γ response in patients vs DQB1*0602 positive controls

A. CD4+ T-cell IFN-γ response to other DQ0602 strong pH1N1 binding peptides

+

+

10

(B) pHA1275-283 peptide were presented using T2.DQ0602 cells and responding CD4+ T

cells from 37 narcoleptic patients (red) and 35 healthy controls (blue) were detected by

IFN-γ ELISpot by counting spot-forming units (SFUs). PMA and ionomycin are used as

positive control.

(C) Dose-response curve for the effect of HCRT56-68, HCRT87-99, and pHA1275-287 on CD4+

T

cell activation, as measured by IFN-γ ELISpot. The epitopes have high affinity for DQ0602

and are highly potent at stimulating CD4+T cells. Data was generated using samples from

5 narcolepsy patients.

11

Figure S7. Representative ELISpot images corresponding to Figs. 4 and 6.

(A) ELISpot results from long-term epitope cross cultures. Cells from a total of 13-14

narcolepsy samples and 3-5 controls were cultured with either mixed HCRT56-68 plus

HCRT87-99, pHA1275-287, or whole vaccine and re-stimulated with HCRT56-68, HCRT87-99,

pHA1275-287, or EBV490-503.

(B) Cross culture stimulation experiments using T2.DQ0602 presentation of HCRT56-68

plus HCRT87-99, pHA1275-287, or EBV490-503 epitopes (24 hr), isolation of CD38+ positive

12

activated cells, and subsequent ELISpot testing for reactivity to HCRT56-68, HCRT87-99,

pHA1275-287, or EBV490-503. In total 14 narcolepsy samples and 8 controls were tested.

Numbers on the bottom right corner of each circle indicate SFU counts for each well.

13

Table S1. Prepro-HCRT peptides showing binding to DQ0602.

The table shows prepro-hypocretin peptides that out-competed a reference peptide by

>90% (very strong binders, SB+), 75-90% (strong binders, SB) or 50-75% (weak binders,

WB). Note that the two very strong binders #15 and #22 are almost identical. a

Shown is

a possible binding motif as predicted by the www.dtu.dk/cbs server.

Prepro-hypocretin

Peptide # Sequence Predicted motifa Binding

1 MNLPSTKVSWAAVTL LPSTKVSWA SB+

6 LLPPALLSSGAAAQP LLSSGAAAQ WB

7 ALLSSGAAAQPLPDC SSGAAAQPL SB

8 SGAAAQPLPDCCRQK GAAAQPLPD WB

10 PDCCRQKTCSCRLYE QKTCSCRLY WB

11 RQKTCSCRLYELLHG TCSCRLYEL SB

12 CSCRLYELLHGAGNH CRLYELLHG SB

13 LYELLHGAGNHAAGI LHGAGNHAA SB

14 LHGAGNHAAGILTLG NHAAGILTL SB

15 GNHAAGILTLGKRRS NHAAGILTL SB+

18 RRSGPPGLQGRLQRL GLQGRLQRL WB

19 PPGLQGRLQRLLQAS GRLQRLLQA WB

20 QGRLQRLLQASGNHA GRLQRLLQA WB

14

21 QRLLQASGNHAAGIL LQASGNHAA SB

22 QASGNHAAGILTMGR NHAAGILTM SB+

23 NHAAGILTMGRRAGA NHAAGILTM SB

26 AGAEPAPRPCLGRRC EPAPRPCLG WB

27 PAPRPCLGRRCSAPA PCLGRRCSA WB

28 PCLGRRCSAPAAASV RRCSAPAAA WB

29 RRCSAPAAASVAPGG PAAASVAPG SB

15

Table S2. pHA1 peptides showing binding to DQ0602.

The table shows pHA1 peptides that out-competed a reference peptide by >75% (SB) or

50-75% (WB). a

Shown is a possible binding motif as predicted by the www.dtu.dk/cbs

server. Sequences were compared to 7 vaccine strains, the A/PR/8/34 strain, and

A/Brevig mission/1/1918 (see also Table S5).

pHA1

Peptide # Peptide sequence Predicted motifa Binding Different from other

H1N1 flu strains

11 VTVTHSVNLLEDKHN THSVNLLED SB No

29 IDYEELREQLSSVSS YEELREQLS WB No

36 PNHDSNKGVTAACPH SNKGVTAAC WB Yes

37 SNKGVTAACPHAGAK SNKGVTAAC SB Yes

38 VTAACPHAGAKSFYK VTAACPHAG WB Yes

39 CPHAGAKSFYKNLIW HAGAKSFYK WB Yes

53 YQNADTYVFVGSSRY QNADTYVFV WB Yes

55 FVGSSRYSKKFKPEI VGSSRYSKK WB Yes

66 ATGNLVVPRYAFAME GNLVVPRYA WB Yes

67 LVVPRYAFAMERNAG YAFAMERNA SB Yes

68 RYAFAMERNAGSGII YAFAMERNA WB Yes

69 AMERNAGSGIIISDT NAGSGIIIS SB Yes

16

70 NAGSGIIISDTPVHD NAGSGIIIS WB Yes

93 HHQNEQGSGYAADLK NEQGSGYAA WB No

94 EQGSGYAADLKSTQN SGYAADLKS WB Yes

95 GYAADLKSTQNAIDE GYAADLKST WB Yes

96 DLKSTQNAIDEITNK QNAIDEITN WB Yes

115 SNVKNLYEKVRSQLK NLYEKVRSQ WB No

129 NREEIDGVKLESTRI IDGVKLEST WB No

132 TRIYQILAIYSTVAS TRIYQILAI WB Yes

133 QILAIYSTVASSLVL STVASSLVL SB No

138 AISFWMCSNGSLQCR MCSNGSLQC SB No

139 WMCSNGSLQCRICI MCSNGSLQC SB No

17

Table S3. pNA1 peptides showing binding to DQ0602.

The table shows pNA1 peptides that out-competed a reference peptide by >75% (SB) or

50-75% (WB). aShown is a possible binding motif as predicted by the www.dtu.dk/cbs

server. Sequences were compared to 7 vaccine strains, the A/PR/8/34 strain, and

A/Brevig mission/1/1918 (see also Table S5).

pNA1

Peptide # Sequence Predicted motifa Binding Different from other

flu strains

6 NLILQIGNIISIWIS GNIISIWIS SB No

7 QIGNIISIWISHSIQ GNIISIWIS SB No

8 IISIWISHSIQLGNQ IWISHSIQL WB Yes

9 WISHSIQLGNQNQIE ISHSIQLGN WB Yes

13 CNQSVITYENNTWVN NQSVITYEN WB Yes

14 VITYENNTWVNQTYV ENNTWVNQT WB No

15 ENNTWVNQTYVNISN WVNQTYVNI SB No

17 TYVNISNTNFAAGQS SNTNFAAGQ WB Yes

18 ISNTNFAAGQSVVSV TNFAAGQSV WB Yes

19 NFAAGQSVVSVKLAG QSVVSVKLA WB Yes

21 VSVKLAGNSSLCPVS AGNSSLCPV WB Yes

29 DVFVIREPFISCSPL IREPFISCS WB No

18

32 SPLECRTFFLTQGAL ECRTFFLTQ WB No

63 GQASYKIFRIEKGKI QASYKIFRI WB Yes

64 YKIFRIEKGKIVKSV IEKGKIVKS WB Yes

90 VWIGRTKSISSRNGF IGRTKSISS WB Yes

105 CIRPCFWVELIRGRP CFWVELIRG WB No

106 CFWVELIRGRPKENT CFWVELIRG WB No

111 SSISFCGVNSDTVGW ISFCGVNSD WB No

19

Table S4. pPB1 peptides showing binding to DQ0602.

The table shows pPB1 peptides that out-competed a reference peptide by >75% (SB) or

50-75% (WB). a

Shown is a possible binding motif as predicted by the www.dtu.dk/cbs

server. Sequences were compared to 7 vaccine strains, the A/PR/8/34 strain, and

A/Brevig mission/1/1918 (see also Table S5).

pPB1

Peptide # Peptide sequence Predicted motifa Binding Different from other

H1N1 flu strains

8 PYSHGTGTGYTMDTV GTGYTMDTV WB No

21 GYAQTDCVLEAMAFL QTDCVLEAM SB No

22 TDCVLEAMAFLEESH LEAMAFLEE SB No

23 LEAMAFLEESHPGIF LEAMAFLEE WB No

26 GIFENSCLETMEVVQ NSCLETMEV WB No

27 NSCLETMEVVQQTRV LETMEVVQQ WB No

34 LNRNQPAATALANTI NQPAATALA SB No

38 VFRSNGLTANESGRL SNGLTANES WB No

40 ANESGRLIDFLKDVM GRLIDFLKD WB No

45 EEIEITTHFQRKRRV IEITTHFQR WB Yes

52 IGKKKQRLNKRGYLI QRLNKRGYL WB Yes

53 KQRLNKRGYLIRALT KRGYLIRAL WB Yes

20

54 NKRGYLIRALTLNTM IRALTLNTM SB Yes

55 YLIRALTLNTMTKDA IRALTLNTM SB No

61 IATPGMQIRGFVYFV TPGMQIRGF WB No

63 RGFVYFVETLARSIC YFVETLARS WB No

65 TLARSICEKLEQSGL SICEKLEQS WB No

83 QPEWFRNILSMAPIM WFRNILSMA WB Yes

84 FRNILSMAPIMFSNK LSMAPIMFS WB Yes

86 PIMFSNKMARLGKGY NKMARLGKG SB No

87 SNKMARLGKGYMFES NKMARLGKG SB No

88 ARLGKGYMFESKRMK LGKGYMFES WB Yes

89 KGYMFESKRMKIRTQ SKRMKIRTQ SB Yes

90 FESKRMKIRTQIPAE SKRMKIRTQ SB Yes

91 RMKIRTQIPAEMLAS QIPAEMLAS SB No

92 RTQIPAEMLASIDLK QIPAEMLAS SB No

97 TKKKIEKIRPLLIDG IEKIRPLLI WB No

98 IEKIRPLLIDGTASL IEKIRPLLI WB No

102 PGMMMGMFNMLSTVL MGMFNMLST WB No

103 MGMFNMLSTVLGVSI NMLSTVLGV WB No

104 NMLSTVLGVSILNLG LGVSILNLG WB No

116 AGVDRFYRTCKLVGI YRTCKLVGI WB Yes

117 RFYRTCKLVGINMSK YRTCKLVGI WB Yes

21

123 TFEFTSFFYRYGFVA EFTSFFYRY WB No

125 YRYGFVANFSMELPS ANFSMELPS WB No

129 GVSGVNESADMSIGV VNESADMSI SB No

130 VNESADMSIGVTVIK NESADMSIG SB No

131 ADMSIGVTVIKNNMI IGVTVIKNN SB No

132 IGVTVIKNNMINNDL IKNNMINND SB No

135 NDLGPATAQMALQLF LGPATAQMA WB No

138 QLFIKDYRYTYRCHR QLFIKDYRY WB No

142 DTQIQTRRSFELKKL DTQIQTRRS WB No

150 NLYNIRNLHIPEVCL NLHIPEVCL SB No

153 VCLKWELMDDDYRGR LKWELMDDD WB Yes

156 RGRLCNPLNPFVSHK CNPLNPFVS WB Yes

159 SHKEIDSVNNAVVMP SVNNAVVMP SB Yes

160 IDSVNNAVVMPAHGP VNNAVVMPA SB Yes

161 NNAVVMPAHGPAKSM NNAVVMPAH WB Yes

166 ATTHSWIPKRNRSIL TTHSWIPKR WB No

167 SWIPKRNRSILNTSQ RNRSILNTS WB No

168 KRNRSILNTSQRGIL NRSILNTSQ WB No

172 DEQMYQKCCNLFEKF QKCCNLFEK WB No

173 YQKCCNLFEKFFPSS QKCCNLFEK WB No

174 CNLFEKFFPSSSYRR NLFEKFFPS WB No

22

177 YRRPVGISSMVEAMV ISSMVEAMV WB No

178 VGISSMVEAMVSRAR SSMVEAMVS SB No

185 KEEFSEIMKICSTIE EEFSEIMKI SB Yes

186 SEIMKICSTIEELRR SEIMKICST WB Yes

187 KICSTIEELRRQK KICSTIEEL SB No

23

Table S5. Influenza A H1N1 strains used for vaccinations by year.

The information is relevant for the northern hemisphere and year 2011 means winter

2010-2011. The information is based on World Health Organization recommendations

for composition of influenza vaccines as curated by www.fludb.org.

* Marks the strains whose sequences were compared to pH1N1 in Tables S2-S5.

Year H1N1 strain selected for vaccinations

2010-2 A/California/07/2009(H1N1)-like virus

2009 A/Brisbane/59/2007(H1N1)-like virus *

2008 A/Solomon Islands/3/2006(H1N1)-like virus

2001-7 A/New Caledonia/20/1999(H1N1)-like virus*

2000 A/Beijing/262/95(H1N1)-like virus*

1999 A/Beijing/262/95(H1N1)-like virus

1998 A/Bayern/7/95(H1N1)-like virus

1988-97 A/Singapore/6/1986(H1N1)-like virus*

1985-7 A/Chile/1/83(H1N1)-like virus*

1981-4 A/Brazil/11/78(H1N1)-like virus*

1979-80 A/USSR/90/77(H1N1)-like virus*

1975-8 None