Alternative Antigen Processing and Presentation Pathways by Tumors - 05

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Cover Page

The handle http://hdl.handle.net/1887/22802 holds various files of this Leiden Universitydissertation

Author : Cunha Oliveira, Claudia daTitle : Alternative antigen processing and presentation pathways by tumorsIssue Date : 2013-12-10

http://hdl.handle.net/1887/22802http://hdl.handle.net/1887/22802http://hdl.handle.net/1887/22802http://hdl.handle.net/1887/22802https://openaccess.leidenuniv.nl/handle/1887/1


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THE NONPOLYMORPHICMHC Qa 1 b MEDIATES

CD8+

T CELL SU RVEILLANCEOF ANTIGEN PROCESSING DEFECTS

Cláudia C Oliveira 1,3, Peter A van Veelen 2,Bianca Querido 1, Arnoud de Ru 2, Sandra Laban 2, Jan W. Drijfout 2, Sjoerd H van der Burg 1,Rienk Offringa 2 and Torbald van Hall 1.

1Depar men o Clinical Oncologyand 2Depar men o Immunohema ology and Blood rans usion,Leiden Universi y Medical Cen er, Leiden, he Ne herlands;3Gradua e Program in Areas o Basic and Applied Biology,Por o, Por ugal

. Offringa and . van Hall con ribu ed equally o his paper

Published in:e Journal o experimen al medicine 2010, 207(1): 207-221.

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ABSTRACTe nonclassical MHC Qa-1 b accommoda es monomorphic leader pep ides andunc ions as a ligand or germ line recep ors CD94/N G2, expressed by na ural killer

cells and CD8+

cells. We here describe ha he conserved pep ides are replaced by anovel pep ide reper oire o surprising diversi y as a resul o impairmen s in he an igenprocessing pa hway. is novel pep ide reper oire represen s immunogenic neo-an igensor CD8+ cells as we ound ha hese Qa-1 b-res ric ed cells dominan ly par icipa edin he response o umors wi h processing deciencies. A surprisingly wide spec rum oarge cells, irrespec ive o rans orma ion s a us, MHC background or ype o processingdeciency was recognized by his cell subse , complying wi h he conserved na ureo Qa-1 b. arge cell recogni ion depended on cell recep or and Qa-1 b in erac ionand immuniza ion wi h iden ied pep ide-epi opes demons ra ed in vivo priming

o CD8+

cells. Our da a reveal ha Qa-1 b

, and mos likely i s human homologueHLA-E, is impor an or he de ense agains processing decien cells by displacing hemonomorphic leader pep ides, which relieves he inhibi ion hrough CD94/N G2Aon lymphocy es and by presen ing a novel reper oire o immunogenic pep ides, whichrecrui s a subse o cy o oxic CD8+ cells.

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INTRODUCTIONQa-1 b and i s human homolog HLA-E are conserved MHC class I like molecules, ofenca egorized as ‘nonclassical’ or class Ib MHC. Similar o he classical MHC molecules, hey

accommoda e small pep ides in heir binding grooves and presen hese on he cell sur ace1, 2

.Crys al s ruc ures o HLA-E show ha he overall old o he pep ide/MHC complex is verysimilar o ha o he classical MHC class I molecules3. e unique ea ure o Qa-1 b and HLA-resides in he very limi ed number o alleles ha is presen in he popula ion4, 5: as ew as woalleles exis in he human popula ion differing jus in one amino acid and his has hardlyany unc ional consequence6. e monomorphic na ure o Qa-1 b and HLA-E is underlined by he nding ha hey are predominan ly lled wi h one pep ide ligand, which is derivedrom he signal sequence o classical MHC class I molecules, ofen re erred o as ‘Qdm’ or‘Qa-1 de erminan modier’7, 8. ese signal pep ides are s rikingly conserved in all es ed

mammalian species9

, poin ing a an impor an unc ion o his pep ide/MHC combina ionor he immune sys em. A subs an ial body o evidence reveals ha i regula es na ural killer(N ) cells and cy o oxic -lymphocy es (C L) via he he erodimeric recep ors CD94/N G2A and CD94/N G2C 2, 10, 11. ese germline-encoded recep ors specically engagehe Qdm pep ide when bound o Qa-1 b/HLA-E, since residues a pep ide posi ion 5 and8 are par o he sur ace in er ace o he CD94/N G2 recep ors12, 13. In eres ingly, hisin erac ion is qui e similar o he oo prin o he hyper variable regions o he cell recep oron pep ide/MHC class I14. Absence o hese Qdm side chain residues resul s in lack orecep or engagemen15, 16 , indica ing ha his inna e recep or is ruly pep ide specic. Qdm/

Qa-1 b

complexes ac as remo e sensors o in egri y o he MHC class I an igen processingmachinery, because de ec s in pro easomal cleavage, AP-media ed pep ide ranspor , signalpep idases or apasin-media ed pep ide loading all resul in ailure o process and presenQdm pep ides17-20. e lack o Qdm/Qa-1 b complexes a he cell sur ace is sensed by N cellsexpressing CD94/N G2 recep ors, and allows or immunosurveillance o such processingdecien cells21. us, his recep or/ligand sys em is a major molecular mechanism behindhe ‘missing sel ’ principle o N cell reac ivi y 22.

In addi ion o heir role in he inna e immune sys em, Qa-1 b and HLA-E serve headap ive response as well. -cell recep or recogni ion o hese nonclassical MHC has

been described in he con ex o immuni y agains in racellular pa hogens, e.g. Lis eria,Salmonella and Mycobac erium uberculosis23-25 , sugges ing ha oreign an igens canreplace he ‘sel ’ Qdm pep ides and be recognized by CD8+ cells. HLA-E-res ric ed cells specic or Qdm do exis , bu have only been repor ed in response o allogeneicMHC26, 27. Fur hermore, Qa-1 b-res ric ed CD8+ cells have been implica ed in heregula ion o experimen al au oimmune diseases (reviewed in21, 28). e an igenic arge so hese regula ory cells are mos likely o ‘sel ’ origin, bu differen rom Qdm. eseda a reveal ha Qa-1 b/HLA-E-res ric ed cells are presen in he hos , bu ha hespecici y, diversi y and unc ion o his -cell subse awai ur her inves iga ion.

Here we describe he exis ence o a surprisingly broad pep ide reper oire ha ispresen ed by Qa-1 b on cells wi h impairmen s in he an igen processing machinery. ese

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CD8+ cell reper oire res ric ed by Qa-1 b


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pep ides replace Qdm and are arge ed by a unique popula ion o CD8+ cy o oxic cells.Normal cells wi h in ac processing machinery were no recognized by hese Qa-1 b-res ric ed C L, bu par ial de ec s readily resul ed in he appearance o he immunogenic‘sel ’ pep ides, which are derived rom housekeeping pro eins. In eres ingly, we showha hese Qa-1 b-res ric ed cells are abundan ly presen in he immune response oprocessing decien umors. Our da a show ha he nonclassical Qa-1 b molecule plays aprominen role in he adap ive immune response as a res ric ion elemen or cells andpresen s a much larger pep ide reper oire han hus ar an icipa ed.

RESULTS

Qa-1 b -restricted T cells are frequently present in the immune responseagainst TAP-deficie nt tumors

We previously described ha he immune response agains AP-decien umorscomprises Qa-1 b-res ric ed cells among cells wi h MHC res ric ion o he classicalclass I molecules29. is promp ed us o de ermine he rela ive con ribu ion o his Qa-1 b-res ric ed subse o he overall response. Mice were immunized wi h syngeneic AP-decien MA-S cells expressing he co-s imula ory molecule CD80. Ex vivo cul uredspleens were examined or IFNγ produc ion agains he paren al MA-S lymphoma, heβ2m-nega ive C4.4-25 line and a panel o single class I expressing EC7.1 cells, which areMHC class I-loss varian s o MA-S30 ( able I). All polyclonalex vivo cul ures respondedo MA-S, bu no agains he β2m-nega ive lymphoma, indica ing ha hey exhibi edspecic -cell reac ivi y agains AP-decien umors, and no merely reec ed na uralkiller (N ) cell reac ivi y. Dissec ion o hese IFNγ responses revealed a co-dominanceo Qa-1 b-res ric ed ac ivi y compared o hose res ric ed by he classical D b- and b-molecules, as he majori y o he examined welve independen cul ures produced highIFNγ levels when incuba ed wi h EC7.1.Qa-1 b ( able I). ese da a sugges ed ha he

Table I. Detection of Qa-1 b-directed IFN γ responses in ex vivo cultures.

Target cells a

RMA-SC4.4-25EC7.1EC7.1.K b

EC7.1.D b

EC7.1.Qa-1 b

EC7.1.controlMedium

# 1 # 2 # 3 # 4 # 5 # 6 # 7 # 8 # 9 # 10 # 11 # 12# 1 # 2 # 3 # 4

6,58282

2167,184

6906,677

250152

# 5

12,199164

1,31116,03110,11316,265

1,370276

# 6

4,89661

7992,5262,1253,1081,065

156

# 7

4,32047

4181,5521,5612,697

40174

# 8

10,3891881

8,309604272

6338

# 9

1,359106485405648

1,410591218

# 10

1,20492

310538645

3,330386287

# 11

2,89423

273441

1,1292,999

418177

# 12

1,5671438

837446

7,0395613

Twelve mice were immunized with RMA-S.B7 and spleens were stimulated twice in vitro with RMA-S.B7 before testing.

a RMA-S is TAP2-deficient; C4.4-25 is b 2m-deficient; EC7.1 is a MHC class I-deficient variant of RMA-S. Single class I geneswere reconstituted in EC7.1.

c Concentration of IFN γ released by ex vivo cultures (pg/ml)

Independent T-cell culture b

97364

5,886888

2,053380287

5,723 c

84712

11,2707,5075,579

700120

10,11361

56411,111

8,3148,827

620110

11,593

b

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nonclassical MHC class I Qa-1 b requen ly ac s as an an igen presen a ion molecule or cells, and even can overrule he classical MHC class I molecules.

Critical involvement of Qa-1 b and the T-cell receptor in target cell recognition

o charac erize hese Qa-1 b-res ric ed cells, we isola ed several -cell clones rom hea oremen ioned cul ures and ex ensively explored he expression and unc ion o sur acerecep ors. All clones displayed a classical C L pheno ype and expressed CD3, CD8αβand C αβ (Fig S1). Differen rearranged C Vβ segmen s were used (no shown), valida ing he independen origin o he clones and indica ing ha Qa-1 b recogni ion doesno cons rain he C reper oire. In addi ion o he -cell lineage recep ors, he -cellclones also expressed CD94, N G2A, N G2C and N G2D, recep ors requen ly oundon Na ural iller (N ) cells, which in erac wi h nonclassical MHC class I molecules,including Qa-1 b. Impor an ly, N lineage markers N 1.1 and DX5, and CD16, CD32,CD244 (2B4) were no expressed, nei her were recep ors o he lec in Ly49 amily, whichin erac wi h classical MHC class I (Fig S1 and da a no shown). We concluded ha ourQa-1 b-res ric ed cells are indis inguishable a he pheno ypic level rom ‘conven ional’CD8+ -cells res ric ed by classical MHC31 , bu are clearly dis inc rom he repor ed Qa-1-recognizing in es inal γδ+ cells32 and Qa-1-res ric ed regula ory CD8αα+ -cell subse33.

o care ully de ermine he Qa-1 b-res ric ion o our isola ed -cell clones, we examinedheir reac ivi y agains wo cell panels, based on he AP-nega ive EC7.1 lymphoma andAP-decien B78H1 melanoma30, 34 (Fig 1). Bo h cell lines are also devoid o MHCclass I pro eins and single class I molecules were recons i u ed by gene rans er. Allisola ed -cell clones exhibi ed cy oly ic ac ivi y (Fig 1A) and produced IFNγ (Fig 1Band Fig S2) agains he Qa-1 b-expressing cells, bu no agains cells in which only D b or b were in roduced. ese da a convincingly demons ra ed he Qa-1 b-res ric ion oour cells and excluded cross-reac ivi y o he classical class I molecules. Qdm-speciccon rol C L did no recognize he AP-decien umor arge s, since he presen a iono Qdm in Qa-1 b is AP-dependen7. Exogenous loading o Qdm con erred reac ivi y orhis C L clone (Fig 1, righ panels). A direc in erac ion wi h Qa-1 b was revealed by hending ha an i-Qa-1 b an ibody s rongly blocked he recogni ion by our C L (Fig 2A).

Nex , we examined he involvemen o an igen-recep ors in arge cell recogni ion. Al hough CD94/N G2A and CD94/N G2C do engage Qa-1 b 2 and were expressed byour isola ed -cell clones, heir involvemen was no likely, because AP-nega ive cellsare devoid o pep ide ligands or hese recep ors35. Indeed, blocking wi h N G2A/C-specic an ibodies did no al er he response, whereas an i-CD3 and -CD8 an ibodiesclearly inhibi ed he recogni ion o EC7.1.Qa-1 b cells (Fig 2B). A role or N G2D wasalso no likely, since our MA-based lymphoma panel lacks N G2D ligands36. oge herhese da a indica ed ha he C governed he reac ivi y o our Qa-1 b-res ric ed C L.

Fur hermore, exogenous loading wi h excessive amoun s o compe ing Qdm pep idesinhibi ed -cell recogni ion o EC7.1.Qa-1 b cells (Fig 2C). Similar compe i ion was ound when he Qdm varian L8 was used (AMAP L L), bu no wi h irrelevan con rol pep ide

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(Fig 2C, lef panel). Qdm-specic con rol C L conrmed ha bo h pep ides are efficien lyloaded on he arge cells (Fig 2C, righ panel). e ac ha he posi ion 8 subs i u ion in hepep ide L8 preven s engagemen o CD94/N G2A recep ors37 , bu re ained he capaci y oinhibi -cell reac ivi y in our assays, subs an ia ed our ndings ha arge cell recogni ion isregula ed by C -media ed in erac ion wi h Qa-1 b and no by N -recep ors.

TAP- and MHC heavy chain-deficiencies are targeted by Qa-1 b-restricted CTL

o evalua e he appearance o he pep ide-epi opes on arge cells ha express na urallevels o Qa-1 b , we es ed B-cell blas s rom spleens o wild ype, AP1-decien and

0

20

40

60

0

20

40

60

0

20

40

60

0

20

40

60

C4.4-25

EC7.1.Qa-1 bEC7.1

EC7.1.Qa-1 b+Qdm

1 : 0 2 1 : 5 1

1 : 0 1 1 : 5

1 : 0 2 1 : 5 1

1 : 0 1 1 : 5

1 : 0 2 1 : 5 1

1 : 0 1 1 : 5

1 : 0 2 1 : 5 1

1 : 0 1 1 : 5

Qa-1 b-restricted CTL Qdm-specific CTL

s i s y l c i f i c e p s t n e c r e P

Effector : Target ratio

A

B

) l m / g n ( e s a e l e r γ N F I

0.0

2.5

5.0

7.5

10.0

12.5

15.0

5 2 - 4 . 4 C

+ D b

+ K b

+ Q a -

1 b

M

e d i u m

B78H1

0.0

2.5

5.0

7.5

10.0

12.5

15.0

5 2 - 4 . 4 C

+ D b

+ K b

+ Q a -

1 b

M

e d i u m

B78H1

0.0

2.5

5.0

7.5

10.0

12.5

15.0

5 2 - 4 . 4 C

+ D b

+ K b

+ Q a -

1 b

M

e d i u m

B78H1

0.0

2.5

5.0

7.5

10.0

5 2 - 4 . 4 C

+ D b

+ K b

+ Q a -

1 b

M

e d i u m

B78H1

+ Q a - 1

b +

Q d m

Figure 1. Selective recognition o Qa-1 b-expressing target cells. Isola ed -cell clones were es edagains panels o AP-decien lymphoma (EC7.1) and melanoma (B78H1) cells. Bo h cell lines are alsodecien in MHC class I30, 34 and were recons i u ed wi h cons ruc s encoding H-2D b , - b or Qa-1 b. (A)Cy o oxic ac ivi y agains Qa-1 b-expressing EC7.1 cells and (B) IFNγ produc ion agains Qa-1 b-expressingB78H1 cells by hree independen -cell clones. Con rol Qdm-specic C L ailed o recognize he Qa-1 b expressing arge s, due o he absence o AP, unless pulsed wi h he Qdm pep ide (righ panels). Meansand s andard devia ions o riplica e wells are shown or one ou o hree comparable experimen s.

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peptide concentration ( µ M)

) l m / g n ( e s a e l e r γ

N F I

0.0

1.0

2.0

3.0

4.0

1 0 1 0 . 1 0 1 . 0

1 0 0 . 0

0.0

2.5

5.0

7.5

10.0Qdm peptideQdm L8K peptide

control peptide

) l m / g n

( e s a e l e r γ

N F I

3 D C - i t n a

4 D C - i t n a

8 D C - i t n a

C / A 2 G K N - i t n a

0.0

2.5

5.0

7.5

10.0

EC7.1.Qa-1 b

E C 7 . 1

-

A

C

B

E C 7 . 1

EC7.1.Qa-1 b

a n t i - Q a -

1

c o n

t . m

A b

-

0.0

2.5

5.0

7.5

10.0

1 0 1 0 . 1 0 1 . 0

1 0 0 . 0

Figure 2. Te -cell receptor, but not NKG2A/C, mediates reactivity o the cells. (A) Blockingan ibody agains Qa-1 b demons ra ed a direc role o his molecule or -cell recogni ion. Experimen was per ormed using EC7.1.Qa-1 b arge cells. (B) Blocking CD3 or CD8 wi h monoclonal an ibodiesdecreased -cell reac ivi y agains AP-decien EC7.1.Qa-1 b arge cells. An ibodies agains N G2A/Cdid no al er he -cell response, indica ing ha only he -cell recep or is cri ically involved inmedia ing reac ivi y. (C) Exogenous pep ide loading compe es wi h endogenously presen ed epi opeson EC7.1.Qa-1 b cells and inhibi s he recogni ion o hese arge cells (lef panel). Qdm (AMAP LLL)or Qdm L8 (AMAP L L) pep ides were loaded exogenously a he indica ed concen ra ions onEC7.1.Qa-1 b cells and IFNγ release by C L was measured. Con rol pep ide was he b-binding 8-merSIINFE L rom OVA. e Qdm L8 mu an pep ide/Qa-1 b complexes ail o in erac wi h CD94/N G2A or CD94/N G2C 37 , indica ing ha he loaded pep ides in er ere wi h -cell recep or media ed

reac ivi y. Qdm-specic con rol C L (righ panel) was ac iva ed by he pep ides. Means and s andarddevia ions o riplica e wells are shown or one ou o hree comparable experimen s.

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β2m-decien mice as arge s or hree independen ly derived C L clones (Fig 3A).AP1-decien cells were efficien ly recognized by all hree clones, bu wild ype andβ2m-decien cells were no . Qdm-specic con rol C L selec ively responded o wildype cells, indica ing ha he absence o Qdm pep ides due o he AP de ec promo edhe presen a ion o he new pep ide-epi opes. Qa-1 b sur ace levels on AP1-deciencells were lower han on wild ype cells, bu were clearly higher han he backgroundlevels on β2m-decien cells (no shown).

We also analyzed C L reac ivi y agains MHC class I heavy chain deciencies wi hhe use o B-cell blas s rom H-2D b- and b-knockou mice. Qdm pep ides are derivedrom he leader sequence o H-2D b 7 and loss o his allele resul ed in recogni ion by wo ohe hree C L clones, whereas deciency in b heavy chains did no resul in recogni ion(Fig 3B). ese sub le differences be ween he C L clones sugges ed ha he cogna epep ides are differen ially inuenced by loss o MHC class I heavy chains. S rikingly, allhree C L clones s rongly responded o cells devoid o bo h alleles (H-2D b b-knockou )(Fig 3B). Con rol blas s lacking β2m in addi ion o H-2D b b ( riple knockou ) were norecognized. ese resul s revealed an unexpec ed inuence o he b molecule on he Qa-1 b-presen ed an igenic pep ides, in ha removal o b molecules rom H-2D b-deciencells s rongly enhanced epi ope display (Fig 3B). e underlying mechanism o hiseffec is curren ly elusive. e degree o C L response o H-2D b b-knockou cells wascomparable o AP-decien cells (Fig 3A), indica ing ha bo h de ec s led o op imalpresen a ion o he new Qa-1 b pep ides. In eres ingly, Qdm-specic C L were able odifferen ia e be ween hese wo arge popula ions, conrming previous ndings ha hisC L clone is also reac ive o AP-dependen pep ides o her han Qdm35, 38. oge her,hese resul s demons ra ed ha our Qa-1 b-res ric ed C L in erac wi h pep ides onac iva ed B-cells wi h de ec s in he an igen presen a ion rou e.

CTL reactivity against tumors with partial processing impairments

We hen de ermined C L reac ivi y agains umor cells wi h AP-dys unc ion andumor cells wi h undened deciencies in he processing machinery. Ad5- rans ormedMouse Embryo Cells (Ad5MEC) and chemically-induced brosarcoma cells (MCA), bo h derived rom AP1-knockou mice, were recognized by he Qa-1 b-res ric edC L (Fig 4A). Correc ion o he AP1 de ec by gene rans er resul ed in decreasedrecogni ion by our C L and s rongly increased recogni ion by he Qdm-specic con rolC L (Fig 4A). us, he al erna ive Qa-1 b-binding pep ide an igens are expressed in a wide array o hema opoie ic and non-hema opoie ic issues o normal and rans ormedcells, including B-cells, broblas s, lymphoma, melanoma and sarcoma, and emerge ahe cell sur ace o cells wi h gene ic disrup ion o he AP pep ide ranspor er.

However, human umors mos ly display par ial deciencies in heir processing pa hwayha origina e a he ranscrip ional level, resul ing in impaired, bu no a comple e shu downin he genera ion o he human coun erpar o Qdm/Qa-1 b complexes. o assess whe herhe C L were also capable o recognize such umor cells, we es ed our chemically-induced

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mouse colon carcinomas, which are known o possess pep ide-presen a ion capaci y 39, 40.Qdm-specic con rol C L indeed recognized all our cell lines, demons ra ing a unc ionalprocessing machinery (Fig 4B, righ panel). In eres ingly, our Qa-1 b-res ric ed C Lresponded o all carcinoma lines, albei o a varying ex en (Fig 4B, lef panel). rea men ohe carcinomas wi h he immunos imula ory cy okine IFNγ, which s rongly improves heprocessing and presen a ion capaci y o cells, resul ed in decreased recogni ion, poin inga par ial processing de ec s in he carcinomas ha were correc ed by IFNγ. In line wi hhis idea, gene rans er o he viral AP-inhibi or UL49.541 led o a s rongly increasedrecogni ion by our C L. As expec ed, he Qdm-specic con rol C L clone exhibi ed heopposi e reac ivi y prole (Fig 4B, righ panel). O no e, he our colon carcinoma lines were derived rom wo mouse s rains wi h differen MHC yping, BALB/c wi h H-2d alleles(C26 and CC36) and C57BL/6 wi h H-2 b alleles (MC38 and CM 93), nicely illus ra inghe conserved na ure o Qa-1, which is he same in hese s rains.

We concluded ha our Qa-1 b-res ric ed C L recognize pep ide-epi opes ha evenappear on he sur ace o cells wi h mild or par ial processing impairmen s, implying haa mix ure o Qdm and AP-independen pep ides can co-exis in Qa-1 b.

AB6

0

5

10

15

0

5

10

15 ) l m / g n ( e s a e l e r γ

N F I

Tap1 –/–

β2m –/–

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5

B

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5

Number of target cells

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5

B6 H-2D b

–/–

H-2D b –/–

K b –/– H-2 K

b –/–

H-2D b –/–

K b –/–

β2m

) l m / g n ( e s a e l e r γ

N F I

0

5

10

15

0

5

10

15

0

5

10

15

0

5

10

15

–/–

0

5

10

15

0

5

10

15

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5

0 0 0 , 0 2

0 0 0 , 5 1

0 0 0 , 0 1

0 0 0 , 5


Figure 3. Qa-1 b-restricted C L are reactive against cells with dened processing deciencies. (A) LPS-s imula ed B-cell blas s rom wild ype (B6), AP1-/- , and β2m-/- mice were used as arge s or Qa-1 b-res ric edC L (lef panels) or con rol Qdm-specic C L (righ panel). (B) LPS-s imula ed B-cell blas s rom wildype (B6), MHC class I-knockou and MHC class I/β2m-knockou mice were es ed or recogni ion byQa-1 b-res ric ed C L (lef panels) or con rol Qdm-specic C L (righ panel). Panels display represen a iveexperimen s ou o our per ormed. Means and s andard devia ions o riplica es are shown.

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Determination of the Qa-1 b -presented peptide repertoire

We se ou o de ermine he na ure o he AP-independen pep ides ha are presen ed by Qa-1 b via biochemical purica ion and andem mass-spec rome ry. Due o lack osui able Qa-1 b specic an ibodies, we made use o a chimeric MHC class I molecule in which he pep ide binding domains (alpha 1 and 2) o Qa-1 b were coupled o he cons analpha 3 domain o D b , agains which good precipi a ing an ibodies are available. ischimeric Qa-1 b/D b molecule was in roduced in o AP2-nega ive EC7.1 cells and inorder o compare he corresponding AP-dependen pep ide reper oire, we also crea ed aAP2-recons i u ed varian o EC7.1.Qa-1 b/D b cells. Flow cy ome ry da a conrmed haEC7.1.Qa-1 b/D b cells only expressed his chimeric molecule on he cell sur ace and nohe endogenous D b molecule, excluding he risk ha we would puri y D b-binding pep ides

C26CC36

CMT93MC38

C26CC36

CMT93MC38

C26CC36

CMT93MC38

IFN g

treated

UL49.5gene

transfer

B

)lm/gn(esaeler γ NFI )lm/gn(esaeler γ NFI

Qa-1 b-restricted CTL Qdm-specific CTLA

0 4 8 120 2 4 6

medium

MCA

Ad5MEC-

+ Tap1

-

+ Tap1

medium

0 1 2 3 4 0 2 4 6

Figure 4. Partial deciencies in the processing pathway o tumor cells induce the novel Qa-1 b-presented peptide-epitopes. (A) Qa-1 b-res ric ed C L respond o Ad5- rans ormed mouse cells(Ad5MEC) and brosarcoma cells (MCA) ha are derived rom AP1-knockou mice. ecogni ion was los upon gene rans er o mouse AP1 in hese cell lines (‘+ AP1’). Qdm-specic con rol C Ldisplayed opposing specici y, indica ing ha he Qdm pep ide is only presen ed on AP-procienumor cells (righ panel). (B) Four colon carcinoma cell lines rom BALB/c (C26 and CC36) orC57BL/6 (MC38 and CM 93) background were used as arge s or bo h C L ypes. Pre- rea men wi h IFNγ o boos he an igen processing and presen a ion machinery resul ed in decreased reac ivi y by he Qa-1 b-res ric ed C L, whereas gene rans er o he viral AP-inhibi or UL49.541 led o s ronglyincreased recogni ion. Again, Qdm-specic con rol C L displayed opposing specici y (righ panel).Means and s andard devia ions o riplica es are shown rom one represen a ive experimen ou o our.

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wi h our alpha 3-direc ed an ibody (Fig 5A). Analysis o AP2 expression in EC7.1.Qa-1 b/D b and in he AP- rans ec ed varian conrmed he absence o AP2 pro ein in hers and success ul recons i u ion in he o her (Fig. 5B). Analysis wi h our Qa-1 b-res ric edC L and Qdm-specic C L conrmed ha our EC7.1 cell lines represen ed wo ex remeso he an igen processing s a us, in ha he AP nega ive line did no presen Qdm andha recons i u ion o AP2 led o a disappearance o he newly dened pep ides and hahe chimeric molecule unc ionally presen ed Qa-1 b-bound pep ides (Fig 5C).

Mass spec rome ry o pep ide species rom puried Qa-1 b molecules demons ra ed aneviden difference be ween he wo pep ide reper oires. e pep ide reper oire o AP-posi ive cells was s rikingly domina ed by he Qdm pep ide AMAP LLL ( able II). Sixo her pep ide sequences were iden ied, bu hese represen ed only minor cons i uen s( oge her less han 5% o he o al ion coun ). In con ras , Qa-1 b rom AP-nega ive cells was lled wi h a remarkable diversi y o pep ides rom endogenous pro eins ( able II). Nopep ide clearly s ood ou in erms o quan i y. More han 150 andem mass ragmen a ionproles sufficien ly ma ched wi h he mouse IPI pro ein da abase (htp://www.ebi.ac.uk/IPI/IPImouse.h ml) and analysis o heir corresponding syn he ic pep ides conrmedhe amino acid sequence o 84 pep ides. e o her pep ide masses could no be ma ched

wi h a known pep ide sequence, bu did no correspond o Qdm or previously denedQa-1 b-binding pep ides2. e leng hs o he AP-independen pep ides ranged rom 8 o18 amino acids, bu he majori y was 9 amino acids long (Fig 5D). Seven pep ides wereound in leng h varian s ( able II, numbers 65 o 84) and six o hese amilies varied aheir C- erminus, reminiscen o leader pep ide processing42. However, only one o heseamilies was ac ually encoded in he leader domain, sugges ing ha amino acid rimminga he C- erminus migh be a ea ure o AP-independen pep ides in general. S rikingly,no clear binding mo i could be elucida ed in our pep ide reper oire, al hough previouss udies wi h syn he ic pep ide libraries showed a dominan role or posi ion 2 (M or L)37.e C- erminus o he pep ides requen ly con ained an alipha ic amino acid (L, I, M, Aand F, see able II), as ound or mos mouse MHC class I alleles.

We concluded ha he presence o he high affini y binding pep ide Qdm in hegroove o Qa-1 b preven s he accommoda ion and display o a broad array o al erna ive

pep ides and ha his new reper oire only emerges upon processing de ec s.Immunogenicity of the Qa-1 b -binding peptides

A direc consequence o he absence o his al erna ive pep ide reper oire underphysiological condi ions is ha his reper oire may comprise neo-an igens or he CD8+ -cell sys em. o es ima e he immunogenici y o he iden ied Qa-1 b-binding pep ides wees ed hem in a ma rix-based screening approach in which each pep ide was sys ema icallyrepresen ed in wo differen pep ide pools con aining ve pep ides each. None o ourQa-1 b-res ric ed -cell clones recognized pep ides rom he iden ied reper oire, sopolyclonal responder cells rom mice immunized wi h AP-decien EC7.1.Qa-1 b.B7 umors were applied, assuring ha he pep ide-direc ed responses were induced by

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8 - m

e r

9 - m

e r

1 0 - m

e r

1 1 - m

e r

1 2 - m

e r

1 3 - m

e r

1 4 - m

e r

1 5 - m

e r

1 6 - m

e r

1 7 - m

e r

1 8 - m

e r0

10

20

30

40

N u m

b e r o f p e p t

i d e s TAP-positive

TAP-negative

Length of eluted peptides

D

AQa-1 b a 1-2

D b a 3

Qa-1/D b:

C

Qa-1/D b

EC7.1

EC7.1.TAP2

Qa-1/D b

-

-

IFN γ release (ng/ml)

100

101

102

103

104

n u m

b e r

RMA

100

101

102

103

104

n u m

b e r

EC7.1

EC7.1. Qa-1/D b

100

101

102

103

104

n u m

b e r

Fluorescence intensity

D b a 3

D b a 3

D b a 3

Qa-1 b

Qa-1 b

Qa-1 b

D b a 2

D b a 2

D b a 2

0.0 2.5 5.0 7.5 10.0 12.5 15.0

Qa-1 b-restricted CTLQdm-specific CTL

TAP2b- actin

B R M A - S

E C 7 . 1

. T A P 2

E C 7 . 1

R M A

- 75 kDa

- 43 kDa

Figure 5. Identication o the AP-independent peptide repertoire o Qa-1 b. (A) e chimericQa-1 b/D b class I molecule was expressed in EC7.1 cells, which are AP- and MHC class I-nega ive. AP-posi ive coun erpar s were genera ed by in roduc ion o he AP2 gene. Sur ace display on ‘EC7.1.Qa-1 b/D b‘ cells o he chimeric molecule and absence o he endogenous D b molecule was de ermined byow cy ome ry wi h, respec ively, Qa-1 b- and D bα3-specic an ibodies, and D bα2-specic an ibody,

as indica ed in he his ogram plo s (clone H95 and also H131-31 (no shown)). S aining o AP-recons i u ed cells gave comparable resul s. Con rol AP-procien MA cells displayed endogenousQa-1 b and D b molecules. (B) AP expression was analyzed on MA, MA-S, EC7.1 and EC7.1. AP2cells by Wes ern blo using he an ibody AP2.688 agains mouse AP2. esul s conrmed he lack oAP2 expression on EC7.1 and MA-S cells. (C) e chimeric cons ruc was recognized by C L clones:he AP-nega ive varian only by Qa-1 b-res ric ed C L (black bars) and he AP-posi ive varian only

by Qdm-specic C L (grey bars). Means and s andard devia ions o riplica e wells are shown rom onerepresen a ive experimen ou o our. (D) Pep ide purica ion and mass spec rome ry analysis revealedhe wide diversi y o he AP-nega ive pep ide reper oire, whereas he AP-posi ive pep ide reper oire

was mainly limi ed o he Qdm pep ide. Da a were collec ed rom our independen experimen s in hecase o AP-nega ive reper oire and wo independen experimen s or AP-posi ive reper oire. Numbero differen pep ides wi h indica ed leng h is depic ed o 84 iden ied pep ides ha are lis ed in able II.

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able II. List o Qa-1 b-binding peptides isolated rom AP-positive and AP-negative tumor cells

Peptidenumber Peptide sequence

PeptideLocation

Proteinlength

UniProtKBor SwissProt

number Protein description

AP-posi ive1 AMAP LLL 3-11 362 P01899 H-2 class I his ocompa ibili y

an igen, D-B alpha chain2 AQAE PEL 691-699 1274 A2 67 DENN domain-con aining

pro ein 33 IIN H LLL 1302-1310 1657 Q80UW7 IQ mo i con aining G Pase

ac iva ing pro ein 14 P FEVID PQS 98-108 108 P97450 A P syn hase-coupling ac or

6, mi ochondrial5 P EEESPV 486-493 493 P53986 Monocarboxyla e ranspor er 16 QAIPQGAIQ 246-254 461 Q8BG99 Homeobox pro ein P NOX2

7 QLQPQQPLPQPQ 125-136 672 Q9WVH4 Forkhead pro ein F H 2AP-nega ive1 AAIENIEHL 1193-1201 1392 Q6PB66 Leucine-rich PP mo i -

con aining pro ein,mi ochondrial

2 AAL LGQEL 799-807 1271 Q9JJ28 Pro ein igh less-1 homologue3 AAP NANSLNS F 454-466 575 Q8B 14 CC 4-NO ranscrip ion

complex subuni 44 AAP SPDHSPA 699-709 709 Q66L44 Pro ein Dos5 AAVIAHDFL 153-161 292 P30282 G1/S-specic cyclin-D36 AGIENDEAF 44-52 248 B1AWD9 Cla hrin ligh polypep ide7 AGPENSS I 383-346 2075 Q80X 6 Au ophagy-rela ed pro ein 2

homologue B8 AGQFNQDYL 45-53 503 Q921F1 Annexin A119 AGV NPQQHL 515-524 636 Q8BN32 Pabpc1 pro ein10 ASLQNFNISNL 2103-2113 2128 B2 J7 Wnk1 pro ein11 SISNPPGSNL 2115-2125 2128 B2 J7 Wnk1 pro ein12 ASQQNSEEM 202-210 210 Q9CXE2 B-cell CLL/lymphoma 7

pro ein amily member A 13 ASVLNVNHI 2195-2203 2603 Q99NH0 Ankyrin repea domain-

con aining pro ein 1714 ASYRQPSVSL 270-280 573 Q62019 16 kD pro ein

15 A PG LIDFL 256-265 648 Q5U222 Ddx5 pro ein16 AVSEG KV Y SA 111-126 126 Q8CGP1 His one H2B ype 1- 17 FAPLP LP L 17-26 156 Q9C 21 Acyl carrier pro ein,

mi ochondrial18 FAPVNVTEV SVE 280-293 462 P10126 Elonga ion ac or 1-alpha 119 FAYEG DYI 137-145 184 Q62143 Qa-2 cell sur ace an igen20 FGPVNHEEL 33-41 147 P46414 Cyclin-dependen kinase

inhibi or 1B21 FQIVNPHLL 634-642 792 Q6NZB3 ibonucleoside-diphospha e

reduc ase22 FQV H VAL 113-121 361 Q9QYA2 Mi ochondrial impor recep or

subuni OM40 homologue

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able II. Continued.

Peptidenumber Peptide sequence

PeptideLocation

Proteinlength

UniProtKBor SwissProt

number Protein description

23 GGPINPAA 1036-1044 1107 Q80X50 Ubiqui in-associa ed pro ein2-like

24 GLGVLLAF 5-12 113 Q8QZ 4 Crumbs pro ein homologue 325 HSIQNSQDM 61-69 210 Q91YN9 BAG amily molecular

chaperone regula or 226 IQ PQIQVY 21-30 119 P01887 Be a-2-microglobulin27 KPPPLPPLVVF 27-38 499 P16277 yrosine-pro ein kinase BL 28 KP NEFYA 190-198 198 O35988 Syndecan-429 CSVSIQVVDV

NDNYPEL328-345 794 Q91XZ8 Pro ocadherin be a 22

30 SAVGHEYV 96-107 486 Q922I8 Hema opoie ic cell specic Lyn

subs ra e 131 LAI NDEEL 93-101 137 Q64426 His one H2A 32 LV PG ALEL 2294-2303 2883 NP_076331 desmoplakin33 ME LA PQ 976-984 1241 B2 XW8 Ppa1 pro ein34 NSI NLD I 100-108 475 P28658 A axin-1035 PADIV NL 12-20 341 Q8VDZ8 Calcium binding pro ein 3936 PD GISSK 52-60 126 Q8CGP2 His one H2B ype 1-P37 PEAFPALA 385-392 392 Q9CY58 Plasminogen ac iva or inhibi or

1 NA-binding pro ein38 PN LVELN 138-146 943 Q6DFV7 Nuclear recep or coac iva or 739 PPSAKAID 97-105 323 Q5SSG6 AA box binding pro ein

( BP)-associa ed ac or40 PP AKAVE 425-433 661 Q5SUS9 Ewing sarcoma breakpoinregion 1

41 PVKVEIEI 155-163 930 Q7 SZ1 Xeroderma pigmen osum,complemen a ion group C

42 SPENPPS EL 171-181 181 Q9CQA0 Cen romere pro ein M43 SPGNSP PM 186-195 469 Q8CI61 BAG amily molecular

chaperone regula or 444 SALINLSSF 7-15 161 Q8VE65 ranscrip ion ini ia ion ac or

FIID subuni 1245 SAPENAV M 28-36 126 O35127 Pro ein C1046 SAPSNFEH 12-20 593 Q8B W9 Serine/ hreonine-pro ein

kinase PA 447 SAP GSG L 227-236 639 Q6P9 1 A P-dependen NA helicase

DDX5148 SAVISLEG PL 156-166 166 P18760 Colin-149 SAVSNNYIQ L 168-178 911 P30999 Ca enin del a-150 SH FSAP 2-10 403 Q3U9L3 ribosomal pro ein L3P amily

member51 SLGINPHVL 966-974 1170 B2 QL0 Nup98 pro ein52 SLG NP DAYL 60-70 172 Q3 HE2 Myosin regula ory ligh chain

M LC253 SQPES VFYL 110-119 245 P63101 Pro ein kinase C inhibi or

pro ein 1

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17/29

A

Immunogenicity score0 5 10 15 20 25 30 35 40

84. KP LFEILNGD83. KP LFEILNG

82. SSPA NISSLEFEDA KLSA81. SSPA NISSLEFEDA KLS

80. SSPA NISSLEFEDA K

79. SSPAN ISSLEFEDA77. LGVKNSEP A

69. AGIENKFGLYLP68. AGIENKFGLYL

67. A GIENKFGL66. AA PRSGPSVA

65. AAP RSGPSV64. YGYSNRVVDLM

63. VTLVNH GSTF62. VSLLNPP ETL

61. VSLLD IDHL60. VQVSNFKSGKGDST L59. VPP VQVSPLIKFGRY

56. TNPESKVFYL55. STIRLLTSL54. SSTTNPKLSTL

53. SQPESKVF YL52. SLGKNP TDA YL

51. SLGINP HVL49. SAVSNNYIQTL48. SAVISLEGKP L47. SAPT GSGKTL

46. SAPSNFEHR45. SAPENA VRM

44. SALINLSSF42. RSPENPPSKEL

41. PVKAVEIEI38. PNKLVELNK35. PAD IVKNLK34. NSIRNLDTI

32. LVRP GTALEL31. LAIRNDEEL

30. KSAVGH EYV29. KCSVSIQVVDVNDNYPEL

26. IQKTP QIQVY24. GLGVLLA F

23. GGPINPA TA22. FQVTHTVA L21. FQIVNP HLL

18. FAP VNVTTEVKSVE17. FAPLPR LPTL15. AT PGRLIDFL

14. ASYRA QPSVSL13. ASVLNVNH I

12. ASQQNSEEM11. KSISNP PGSNL10. ASLQNFNISNL9. AGVRNPQQHL

8. AGQFNQDYL6. AGIENDEA F5. AA VIAHD FL

3. AA PTN ANSLNSTF2. AALKLGQEL

1. A A IENIEHL

Figure 6. Immunogenicity o Qa-1 b-presented peptides in vitro and in vivo. (A) Immunogenici y o59 pep ides was de ermined by compiling he IFNγ response o wen y-ve -cell cul ures, which wereinduced by immuniza ion wi h irradia ed AP-nega ive EC7.1.Qa-1 b.B7 cells. esponses were measuredusing a ma rix-based approach in which each pep ide was represen ed in wo independen pools. -cell

responses agains each pool were scored rom zero o hree and compiled scores rom he wo pools weremul iplied. (B-D) Percen age o IFNγ producing CD8+ -cells in he blood o mice ha were naïve

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% s

p e c i

f i c

I N F g - p o s

i t i v e C D 8 +

3. A A P

T N A N S

L N S T

F

1 2. A S

Q Q N S

E E M

1 7. F A

P L P R L

P T L

2 9. K C

S V S I Q

V V D V

N D N Y

P E L

Q d m

4 1. P V K

A V E I E

I

0.0

0.5

1.0

1.5

2.03.04.05.0

B

C

D

E

F

G

Naive

Immunized

medium peptide 17 peptide 17Qdm

0

20

40

60

80

3. A A P

T N A N S

L N S T

F

1 2. A S

Q Q N S

E E M

1 7. F A

P L P R L

P T L

2 9. K C

S V S I Q

V V D V

N D N Y

P E L

Q d m

4 1. P V K

A V E I E

I

% s

p e c i

f i c

k i l l i n

v i v

o

10 0 10 1 10 2 10 3 10 4 1 0

0

1 0

1

1 0

2

1 0

3

1 0

4

100

101

102

103

104

1 0

0

1 0

1

1 0

2

1 0

3

1 0

4

I N F γ

CD8

I N F γ

CD810

010

110

210

310

410

010

110

210

310

4

c o u n

t s

c o u n

t s

fluorescence fluorescence

0.03 0.06

0.25 1.12

no pepQdm

Qdm

no pepQdm

pep

no pepQdm

Qdm no pep Qdm

pep

Figure 6. Continued. (B) or immunized wi h he indica ed pep ide (C). Blood samples were aken andcells were s imula ed overnigh wi h medium or specic pep ide, s ained wi h an ibodies and analyzed byow cy ome ry. Collec ed da a rom pep ide-specic requencies o CD8+ cells rom mice are depic edin (D). Each da a poin represen s one mouse, and da a rom wo independen experimen s is shown.(E-G) Cy o oxic reac ivi y in vivo in he same mice as shown in (D). Specic killing was de ermined innaïve (E) or immunized mice (F) by comparing he numbers o CFSEhigh arge s, which were loaded

wi h relevan pep ides, o CFSElow

arge s, which were no loaded wi h pep ide. CFSEin ermedia e

arge s were loaded wi h Qdm and were always comparable o arge s wi hou pep ide. e means o percen agein vivokilling is depic ed wi h s andard devia ions (G).

na urally presen ed an igens. esponses o wen y-ve independen -cell cul ures werecompiled and nal immunogenici y scores revealed a number o immunogenic pep ides(Fig 6A). ese pep ide an igens had no common amino acid mo i and were derived romdifferen housekeeping pro eins (see corresponding numbers in able II), again poin inga he broad diversi y o his an igen reper oire. o assess he po ency o he ve mosimmunogenic pep ide sequences (number 3, 12, 17, 29 and 41) o induce CD8+ cell

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responsesin vivo , we immunized mice wi h he pep ides in conjunc ion wi h imiquimodas adjuvan . Afer wo vaccina ions, clear popula ions o pep ide-specic CD8+ cells were de ec able in he blood (Fig 6B-D). e pep ides wi h he larges cell popula ionsalso scored high in he immunogenici y screen in which umor-s imula ed cells wereapplied (Fig 6A). In eres ingly, pep ide immuniza ions in H-2D b-/- b-/- mice did no leado a popula ion o IFNγ-posi ive CD8+ cells, indica ing ha he cell reper oire in heseknockou mice is oleran or he novel Qa-1 b binding pep ides (Fig S3). is is in line wi h he nding ha H-2D b-/- b-/- arge cells efficien ly presen ed hese pep ides (Fig 3B), which are par o he normal Qa-1 b pep ide reper oire ha induces cen ral olerance in hehymus. Impor an ly, he vaccine-induced CD8+ cells in wild ype mice exhibi ed s rongcy oly ic reac ivi yin vivo agains arge cells ha were loaded wi h he relevan pep ide(Fig 6E-G). Unloaded arge cells and cells loaded wi h Qdm pep ide were no killedinvivo. Vaccina ion wi h he Qdm pep ide did no rigger any -cell immuni y, illus ra ingha he -cell reper oire or his sequence does no exis or is unc ionally oleran .

In conclusion, a broad reper oire o Qa-1 b-binding pep ide-epi opes o ‘sel ’ originemerges a he sur ace o cells wi h impairmen s in he an igen processing pa hway and hahis novel reper oire replaces he AP-dependen Qdm. ese resul s underscore a role oQa-1 b in adap ive immuni y and in er ha cell responses are evoked via his nonclassicalMHC molecule o preven ou grow h o processing-decien cells in he body.

DISCUSSION

e nonclassical MHC class I molecule Qa-1 b normally presen s monomorphic Qdmpep ides derived rom he leader sequences o classical MHC. is Qdm pep ide/MHCcomplex unc ions as a remo e sensor o he class I processing pa hway in egri y hroughde ec ion by CD94/N G2A recep ors on N cells. Failure o presen Qdm, due oimpairmen s in pro eolysis, AP or o her means, increases he suscep ibili y o cells oratack by hese lymphocy es. We here show ha Qdm under such condi ions is replaced bya surprisingly broad reper oire o al erna ive ‘sel ’ pep ides. is novel pep ide reper oirecomprises neo-an igens or which he -cell sys em is no hampered by immunologicalolerance and can here ore induce a vigorous response o Qa-1 b-res ric ed CD8+ C L.is an igen presen ing unc ion o Qa-1 b places his conserved MHC molecule a hein ersec ion o inna e and adap ive immuni y. On one hand, i plays a role as a monomorphicinhibi ing ligand when Qdm is presen ed, and, on he o her hand, as a display sys em oan immunogenic ‘sel ’ pep ide reper oire. Our da a sugges ha he immune sys em isequipped wi h mul iple mechanisms o remove processing decien cells rom he body.

e nding ha Qa-1 b is capable o presen ing o her pep ides han Qdm is suppor ed by previous work rom several groups. e leader o he CMV pro ein UL40 was oundo be presen ed, as well as pep ides romSalmonella , Listeria , EBV and Mycobacterium

tuberculosis2. Fur hermore, an increase o Hsp60 in he cellular s ress response was showno resul in replacemen o he human Qdm homolog wi h he Hsp60 signal pep ide43.

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e unique ea ure, however, o he pep ide reper oire we have elucida ed in our curren work is he ac ha hese pep ides are derived rom endogenous housekeeping pro einsand only emerge in Qa-1 b on he cell sur ace when he processing pa hway is hampered. We demons ra ed he display o hese pep ides in cells wi h par ial and mild decienciesha do no comple ely preven Qdm rom appearance, bu a comple e block in heprocessing pa hway, e.g. loss o AP1 or AP2, resul ed in he mos efficien presen a iono he novel pep ides, demons ra ing ha AP-independen processing mechanisms areresponsible or he replacemen o Qdm. Surprisingly, cell sur ace expression o Qa-1 b is less affec ed by AP deciency han classical MHC class I molecules44 , and HLA-Esur ace expression has also been described in he absence o unc ional AP pro eins orclass I-derived signal pep ides45, 46. Qa-1 b and HLA-E, indeed, seem o efficien ly egressrom he E in he absence o AP47, 48. ese da a implica e ha our mouse s udies arerelevan or he human seting. wo al erna ive processing rou es have been described by which pep ides can bypass he AP ranspor er: N- erminal signal pep ides, whichare processed by signal pep ide cleaving enzymes42 and C- erminal ends o E -residenpro eins can also be loaded wi hou AP unc ion, al hough he responsible pro eoly icenzymes have no been iden ied or his pa hway 49. For y percen o he 84 iden iedpep ides in our search was indeed loca ed a he N- or C- erminus. In eres ingly, one o hemos immunogenic Qa-1 b pep ide-epi opes FAPLP LP L is encoded in he N- erminalsignal sequence o a mi ochondrial carrier pro ein (UniPro Q9C 21). e ac hasix y percen o he pep ides canno be explained by hese wo processing mechanismssugges s he exis ence o o her ye uncharac erized processing pa hways or MHC classI an igen presen a ion. I has been proposed ha au ophagy migh con ribu e o MHCclass I-res ric ed an igen presen a ion50. Au ophagy is a crucial cellular mechanismresponsible or he clearance o old or damaged cellular componen s, includingorganelles and macromolecules. is pa hway migh lead o he genera ion o pep idessuscep ible or MHC class I loading. Previous charac eriza ions o pep ides bound oclassical MHC class I rom AP-decien cells revealed similar ea ures: predominanceo signal pep ides, increased pep ide leng hs, and selec ive presen a ion on AP-nega ive varian s51, 52. e Qa-1 b pep ide reper oires presen on AP-posi ive versus AP-nega ivecells were ex remely differen ( able II), in ha he high affini y binding monomorphicQdm s rongly domina ed he AP-posi ive pool or more han 95%, whereas a broaddiversi y o o her pep ides was presen in he AP-nega ive pool. Previous comparisonso pep ide reper oires presen ed by classical MHC class I molecules showed much moreoverlap be ween he AP-posi ive and -nega ive pools51, 52. is indica es ha he highaffini y binding Qdm compe es wi h he AP-independen pep ides and hereby preven sheir presen a ion under normal circums ances. However, even mild or par ial processingdeciencies in umor cells already led o he presen a ion o bo h ype o pep ides, asillus ra ed by he ac ha Qdm-specic C L as well as our Qa-1 b-res ric ed C L wereable o recognize he our colon umors (Fig 4). Impor an ly, Qdm is no immunogenicdue o he widespread presen a ion in he normal hos , including hymus53 , and hereby

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preven olerance mechanisms or C L specici ies ha recognize he new emergingpep ide-epi opes. We indeed ound ha cells specic or hese novel Qa-1 b pep idesare dele ed in mice ha do presen hese pep ides in Qa-1 b molecules during -celldevelopmen , as in H-2D b-/- b-/- mice (Fig S3).

Our Qa-1 b-res ric ed -cells exhibi a normal pheno ype or CD8+ -lymphocy es(Fig S1) and use rearranged αβ C s and hereby dis inguish hemselves rom repor edQa-1-recognizing in es inal γδ+ -cells32 and he Qa-1-res ric ed regula ory CD8αα+ -cell subse 33. arge cell recogni ion was media ed by he C and his recep or did nocross-reac wi h classical class I molecules, indica ing ha also a he unc ional level hissubse is no differen rom conven ional CD8+ -lymphocy es. is is in line wi h da arom mice lacking classical MHC class I molecules arguing ha approxima ely 10% o heo al CD8+ -cell subse is res ric ed by nonclassical molecules23, 54-56. is cell reper oireis he erogeneous, as wi nessed by a diverse usage o C Vβ segmen s54, 56. Selec ion oQa-1 b-res ric ed -cells in he hymus indeed does occur53 , bu i remains puzzling howa monomorphic Qdm/Qa-1 b complex can media e posi ive selec ion resul ing in such a broad reper oire. Fur hermore, he rela ionship be ween he suppressive Qa-1 b-res ric ed-cells 21, 28 and he umor-reac ive -cells described here remains elusive.

We specula e ha comparable -cell subse s exis in human beings, especially becauseQa-1 b and HLA-E are ra her conserved in s ruc ure and unc ion2. C -media edrecogni ion o HLA-E has been described25-27 and i is highly likely ha a similar subse asour Qa-1 b-C L, which responds agains umors wi h processing impairmen s, is presenin humans. HLA-E sur ace expression is ubiqui ous in he body and even de ec ed inhe absence o he human Qdm homolog45, 46 , al hough MHC class I leaders s ronglys imula e HLA-E display 19, 57. e lack o polymorphism in he human popula ion in ersha iden ied neo-an igens presen ed in HLA-E cons i u e universal epi opes ha migh

be exploi ed or he herapy o requen ly occurring umor immune escape varian s andpersis en in ec ions by viruses encoding immune evasion pro eins.

MATERIAL AN D METHODS

Cell lines and mice

e origin and cul uring o mos cell lines used in his s udy was described previously 29.C4.4-25- is a β2m decien varian o EL4. MA-S.B7-1 is a CD80 rans ec an oMA-S. EC7.1 is a b- and D b-nega ive varian o MA-S30. B78H1 is a AP-decienand MHC class I decien melanoma34. ap1 –/– and wild ype mouse embryo broblas s were immor alized by he adenovirus ype 5 E1 gene (Ad5MEC, clone XC3). ap1–/– brosarcoma was induced wi h me hylcholan rene (MCA, clone MCB6 AP). Generans er o mouse AP1, mouse AP2, H-2D b , H-2 b and Qa-1 b ( 23 gene) wasper ormed wi h re roviruses based on he MuLV vec or LZ S. Genera ion o C L

clones was per ormed according o previous descrip ion29

. Briey, syngeneic C57BL/6mice were immunized wi h irradia ed MA-S.B7-1 cells and in vi ro s imula ed weekly

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wi h a mix ure o MA-S.B7-1 and EC7.1.Qa-1 b.B7-1 cells, naïve splenocy es and IL-2.Qa-1 b-res ric ed C L clone B12i ha is reac ive wi h he AP-dependen AMAP LLLpep ide was isola ed rom a B6. la mouse38.

All mice were purchased rom Iffa Credo (France) and housed in he animal acili yo he Leiden Universi y Medical Cen er under specic pa hogen- ree condi ions andused be ween 8 and 12 weeks o age. AP1-knockou mice were purchased rom Jacksonlabora ories and MHC class I-knockou animals were ob ained rom Dr. F. Lemonnier56.Experimen s were per ormed in accordance wi h Du ch na ional legisla ion andins i u ional guidelines and were approved by he local e hical commitee.

CTL assays and flow cytometry C L ac ivi y was measured by chromium (51Cr) release assay or IFN-γ ELISA as described be ore58. Da a shown represen mean values ob ained rom riplica e es -wells and he

error bars represen s andard devia ion o hese values. For an ibody blocking, C L werepre- rea ed wi h 20 µg/ml an ibodies, washed and added as responders o arge cells orIFN-γ release. e ollowing an ibodies were used in unc ional blocking assays: hams eran i-CD3 (Fab2 ragmen s o clone 145-2C11), con rol hams er an i- NP, ra an i-CD4(clone G 1.4), ra an i-CD8 (clone 2.43) an i-N G2A/C (clone 20d5) and an i-Qa-1 b (clone 6A8.6F10.1A6). For ow cy ome ry analysis, he ollowing monoclonal an ibodies were purchased rom Bec on Dickenson: Ly49A B6 (clone A1), Ly49C/I (clone 5E6),Ly49G (clone LGL-1), Qa-1 b (clone 6A8.6F10.1A6), N G2A B6 (clone 16a11), CD94(clone 18d3), CD3 (clone 145-2C11), CD8α (clone Ly2), CD8β (clone Ly-3.2.), CD4

(clone G 1.4), CD16/CD32 (clone 2.4G2), N 1.1 (clone P 136), CD49b (cloneDX5), CD244 (clone 2B4). From eBioscience we purchased N G2D (clone CX5).

Peptide elution, HPLC and mass spectrometry

Pep ides were elu ed ou o puried chimeric Qa-1 b/D b molecules rom EC7.1 cells. egenomic H-2D b cons ruc in which he alpha 1 and alpha 2 domains were exchanged wi hhose o Qa-1 b was kindly provided by Dr. PJ Dyson (Uni ed ingdom)38. is genomiccons ruc was expressed in HeLa cells and cDNA was hen cloned in o he re roviralcons ruc LZ S. EC7.1 cells ransduced wi h his cons ruc were wice sor ed by FACS.

AP-posi ive coun erpar s were genera ed by re roviral gene rans er o he mouse AP2gene. Immunoprecipi a ion o Qa-1 b/D b molecules was per ormed wi h pro ein A beadscovalen ly coupled wi h an i-D b mAb 28-14-8S rom wo and our independen lysa es o40.109 AP-posi ive and AP-nega ive EC7.1 cells, respec ively, as previously described58.e pep ide pools were pre rac iona ed on a C18 P-HPLC sys em (200 µm x 15 cm;eprosil-C18-AQ 3 µm (Dr. Maisch GmbH, Ammerbuch, Germany)). Frac ions werereduced o near dryness, dilu ed in 95/3/0.1 v/v/v wa er/ace oni ril/ ormic acid andsubsequen ly analyzed by andem mass spec rome ry. Pep ides were analyzed by nanoowliquid chroma ography using an Agilen 1100 HPLC sys em (Agilen echnologies),

as previously described29

, coupled on line o a 7- esla L Q-F mass spec rome er( ermo Elec ron, Bremen, Germany). e end o he nanocolumn was drawn o a ip

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(ID approxima ely 5 µm), rom which he eluen was sprayed in o he mass spec rome er.Pep ides were rapped a 5 µl/min on a 1-cm column (100-µm ID, eproSil-Pur C18-AQ,3 µm) and elu ed o a 15 cm column (50-µm ID, eproSil-Pur C18-AQ, 3 µm) a 150 nl/minin a 60-min gradien rom 0 o 50% ace oni rile in 0.1% Formic acid.

e mass spec rome er was opera ed in da a-dependen mode, au oma ically swi ching be ween MS and MS/MS acquisi ion. Full scan MS spec ra were acquired in he F -IC wi h a resolu ion o 25,000 a a arge value o 3,000,000. e wo mos in ense ions were hen isola ed or accura e mass measuremen s by a selec ed ion moni oring scan inF -IC wi h a resolu ion o 50,000 a a arge accumula ion value o 50,000. e selec edions were hen ragmen ed in he linear ion rap using collision-induced dissocia ion aa arge value o 10,000. In a pos analysis process, raw da a were conver ed o peak lis susing Bioworks Browser sofware, Version 3.1. For pep ide/pro ein iden ica ion, MS/MS da a were submited o he mouse IPI da abase using Masco Version 2.1 (Ma rixScience) wi h he ollowing setings: 5 ppm and 0.8-Da devia ion or precursor andragmen masses, respec ively; no enzyme was specied. All pep ides wi h a masco ionscore above 30 were manually inspec ed. is resul ed in an ini ial se o 150 pep ides.ese pep ides were syn hesized and heir MS/MS spec ra compared wi h he MS/MSspec ra o he Qa-1 b-elu ed pep ides. is resul ed in 84 conrmed pep ide iden ica ions.

Immunogenicity screen of Qa-1 b presented peptides

C57BL/6 mice were immunized wice wi h irradia ed EC7.1.B7-1 cells wi h s ableexpression o Qa-1 b. Spleens o immunized mice were cul ured wi h he same cells or

one o wo weeks and reac ivi y o he cul ures was es ed agains he EC7.1 cell panelo conrm Qa-1 bres ric ion o he -cells. eac ivi y agains he iden ied pep ides wasassessed using a ma rix-based sys em wi h pools o 5 differen pep ides and in which eachpep ide was represen ed in wo independen pools. e pep ide pools were loaded onspleen cells and IFNγ produc ion by -cell was measured wi h sandwich ELISA afer 18 h.eac ivi y agains each pool was arbi rarily scored rom zero o hree, according o hemean background response. wen y ve independen cul ures were assayed in his wayand he scores o each pool compiled. e nal immunogenici y score or each pep ide washen ob ained by mul iplying he scores o wo pools con aining ha par icular pep ide.

Peptide immunizations and in vivo cytotoxicity assay

For pep ide immuniza ions, C57BL/6 and H-2D b-/- b-/- mice were injec ed s.c. a heank wi h 50 µg pep ide mixed wi h 50 µg helper pep ide rom MuLV (H1929) in PBS onday 0 and 7. Immedia ely ollowing injec ion, 60 mg Aldara™ cream (3M Pharmaceu ical)con aining 5% imiquimod was applied o he skin a he injec ion si e. Mice received i.p.injec ions o 500,000 IU recombinan human IL-2 (Novar is) on he day o he second vaccina ion and on he day hereafer. -cell requencies were de ermined rom bloodlymphocy es afer our days and rom spleens afer seven days wi h in racellular cy okine

s aining, as previously described59

. In shor , cells were cul ured overnigh wi h mediumor 5 µg/ml o pep ide and s ained or CD8 and in racellular IFNγ. illing capaci y o

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or PA28. J.Exp.Med. 192 , 483-494 (2000).59. Bijker, M.S. e al. CD8+ C L priming byexac pep ide epi opes in incomple e Freund’sadjuvan induces a vanishing C L response, whereas long pep ides induce sus ained C Lreac ivi y. J Immunol 179 , 5033-40 (2007).

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B6 mice D bK b-/- mice

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Figure S2. Selective recognition o Qa-1 b-expressing target cells. Isola ed -cell clones were es edagains a panel o AP-decien lymphoma (EC7.1) cells. EC7.1 cells are also decien in MHC class I30 and were recons i u ed wi h cons ruc s encoding H-2D b , - b or Qa-1 b. IFNγ produc ion agains Qa-1 b-posi ive EC7.1 cells by hree independen -cell clones. Con rol Qdm-specic C L ailed o recognizehe Qa-1 b expressing arge s, due o he absence o AP. AP-posi ive lymphoma cells ( MA) wererecognized by his C L clone (righ panel). Means and s andard devia ions o riplica e wells are shownor one ou o hree comparable experimen s.

Figure S3. Immunogenicity o Qa-1 b-presented peptides in MHC class I knockout mice. H-2D b-/- b-/- and con rol B6 mice were immunized wi h wo AP-independen pep ides FAPLP L L (pep ide17) and CSVSIQVVDVNDNYPEL (pep ide 29) and Qdm. PBL rom blood samples were incuba edovernigh wi h selec ed pep ides (panel A) or PMA/Ionomycin mi ogens (panel B) and in racellularIFNγ produc ion in CD8+ cells was de ermined by ow cy ome ry. Frequencies o IFNγ producing

-cells were observed agains Qa-1 b

pep ides in con rol B6 mice bu no in MHC class I knockou mice.PMA induced ac iva ion o cells rom all mice. Each da a poin represen s one mouse.

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