Alternative Antigen Processing and Presentation Pathways by Tumors - 02

8/9/2019 Alternative Antigen Processing and Presentation Pathways by Tumors - 02

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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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PEPTI DE TRANSP ORTER TAP

MEDIATES BETWEEN COMPETING

ANTIGEN SOURCES GENERATINGDISTI NCT SURFACE

MHC CLASS I PEPTIDE REPER TOIRES

Cláudia C. Oliveira1,3, Bianca Querido1*, Marjolein Sluijter1*, Jens Derbinski2,Sjoerd H. van der Burg 1 and Torbald van Hall1

1Deparmen o Clinical Oncology,Leiden Universiy Medical Cener, he Neherlands;2Division o Developmenal Immunology, umor ImmunologyProgram, German Cancer Research Cener, Germany;3Graduae Program in Areas o Basic and Applied Biology,Universiy o Poro, Porugal

* Tese auhors conribued equally o his work

Published in:European journal o immunology 2011, 41(11): 3114-3124."In his Issue" European Journal o Immunology 2011, 41: 3092–3093

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| PEPTIDE TR ANSPORTER TAP PREVEN TS PRES ENTATION OF

ALTERNATIVE PE PTIDES

Te pepide ransporer AP mediaes he enry o pepide precursors rom he cyosol

ino he ER where hey are loaded ino MHC-I molecules. Par o he MHC-I presenedpepides do no require he acion o AP or he proeasome and derive rom alernaive

processing pahways. Ineresingly, some o hese alernaive pepides are only presened

when here are impairmens in he classical processing pahway and do no reach he

cell surace in normal cells. In his issue, Oliveira et al now describe ha he pepide

ransporer AP acually prevens he presenaion o his alernaive reperoire due ohe overwhelming influx o compeior pepides in he ER. Srong over-expression o

he anigen-encoding gene was needed o push he alernaive pepide owards MHC-I

surace display. Tus, AP behaves like a lever o conrol o shi he presened pepide

reperoire gradually owards AP-independen or AP-dependen pepides.

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| ABSTRACT

We recenly described a caegory o AP-independen pepide-epiopes ha are selecively

presened by cells wih processing deecs in he classical MHC class I (MHC-I) pahway.

Here, we sudied he ER-residen ceramide synhase rh4 as a prooypic example o heseneo-anigens and ound ha moderae inhibiion o AP permis cell surace presenaion

o he rh4 pepide. Te absence o his pepide rom W cells was no relaed o he

binding or sabiliy o he rh4/D b complexes, or o he availabiliy o MHC-I heavy

chains, bu raher o he limied expression o he anigen. Srongly elevaed anigen levels

were needed o reach comparable pepide display on W as on AP-deficien cells. Our

daa sugges ha he normal influx o AP-ranspored pepides in he ER during rouine

processing creaes an efficien barrier or pepides rom alernaive processing roues.

Impairmen o AP uncion, as commonly ound in cancers and virus-ineced cells,

lowers his resisance allowing or MHC-I presenaion o oher pepide sources.

| INTRODUCTION

Cyooxic lymphocyes (CLs) are key effecor cells o he adapive immune sysem

and circulae hroughou he body in search or heir cognae pepides ha are presened

by MHC class I (MHC-I) molecules. -cell recepors deermine he anigen specificiy

o CLs and engagemen wih pepide/MHC-I complexes leads o heir acivaion

and eliminaion o arge cells. Tereore, he process o MHC-I anigen processing

and presenaion, which operaes in all nucleaed cells o our body, is crucial or CL

immune surveillance1-3. Te highly complex reperoire o MHC-I presened pepides

reflecs he oal proeome o cells and derives rom physiological urnover o proeins,

a process ha is largely operaed by he mulicaalyic enzyme proeasome4, 5. In addiion

o he proeasome, oher proeolyic enzymes in he cyosol have been implicaed in he

liberaion o pepides or MHC-I presenaion, some o which can compensae or he lack

o proeasome aciviy 1, 6, 7. For insance, ripepidyl pepidase II (PPII), insulin-degrading

enzyme (IDE), hime oligopepidase (OP) and nardilysin have been implicaed in he

generaion o some CL epiopes8-10. However, he relaive conribuions o hese novel

pepidases and heir cooperaion wih he proeasome have no been ully characerized.

Te inermediae pepide producs are rescued rom oal breakdown by hese

cyosolic proeases hrough ranslocaion ino he ER. Subsequenly, pepides are

rimmed and loaded ino he grooves o MHC-I molecules, a dynamic process ha is

mediaed by he pepide loading complex (PLC) consising o MHC-I, β2m, ERp57, AP,

apasin and chaperones11-13. Te AP pepide ranspor is operaed by he heerodimer

pump AP1/AP2, members o he ABC ransporer amily. Te imporance o he AP

ransporer is highlighed by he srong decrease in cell surace MHC-I molecules in

he absence o AP14. Furhermore, his botleneck o he anigen processing pahway

is requenly argeed by viruses, especially rom he herpes group, which successully

evade complee eradicaion by CL immuniy 15, 16. Moreover, loss o AP expression

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Pepide ransporer AP mediaes beween compeing sources o MHC-I pepides


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is oen ound in cancers and resuls in resisance o CL atack 17-19. All hese findings

ogeher convincingly demonsrae ha he convenional proeasome-AP pahway plays

a dominan role in he surace display o pepide/MHC-I.

In addiion o he convenional MHC-I processing pahway described above, cells

are equipped wih alernaive roues ha lead o he liberaion and loading o pepides

in MHC-I molecules. Te exac conribuion o hese alernaive roues o he overall

pepide reperoire is difficul o assess, bu i is inriguing ha AP-knockou mice

sill harbor a raher diverse CD8+ -cell reperoire ha can uncionally respond o

viral anigens14, 20. Moreover, AP-deficien paiens seem o cope quie well wih viral

inecions, in conras o inecions wih Gram-negaive baceria21-24. Apparenly, AP-

independen processing pahways can parly compensae or he loading o pepides in

MHC-I molecules. Biochemical analysis o he pepide reperoire o AP-deficien 2

cells revealed abundan presenaion o signal sequence derived pepides25-27. Indeed,

signal sequences are liberaed by he combined acion o signal pepidase (SP) and signal

pepide pepidase (SPP) and are direcly available or loading ino MHC-I28, 29. A second

characerized processing pahway ha bypasses AP is acive in he secreory roue and

is mediaed by members o he proproein converase (PC) amily, like urin30-32. Tis

enzyme is locaed in he rans-Golgi nework and mediaes he proeolyic maurarion o

many proproeins, e.g. growh acors and marix mealloproeinases33. Pepides locaed

a he C-erminus o secreed proeins can be liberaed by urin and subsequenly gain

access o MHC-I in a AP-independen way 30, 32.

Previously we repored he AP-independen presenaion o a C-erminal pepide

rom he ceramide synhase rh4, which is a muliple membrane-spanning proein in he

ER 34-36. Tis pepide was he firs naural example o a C-erminal processing pahway o

ER residen proeins, while previous sudies suggesed he exisence o his roue37-39. Te

rh4 proein has a housekeeping uncion and is ubiquiously expressed. Inriguingly,

he C-erminal pepide-epiope was no presened by processing-inac cells, bu only

emerged in MHC-I o cells wih processing deecs, like proeasome, apasin or AP36.

In our curren sudy, we show ha pepide reperoires rom he convenional processing

roue and he AP-independen roue are shiing arrays conrolled by AP uncion. Te

AP-mediaed pepide influx in he ER seems o consiue a resisance barrier or he

presenaion o pepides rom alernaive processing roues.

| RESULTS

Selective presentation of self-peptide Trh4 by processing-deficient cells

Te surace presenaion o he rh4 pepide was deermined or wo panels o umor

cells using previously esablished CL clones. RMA-S lymphoma cells are AP-deficien

due o an incomplee AP2 chain and he rh4 pepide, bu no a proeasome- and AP-

dependen conrol pepide, was deeced a he surace o hese cells (Fig 1A). Vice versa,

AP-inac RMA cells efficienly presened he conrol epiope, bu he rh4 pepide

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was no deeced by he CL clone (Fig 1A). In he fibrosarcoma umor model MCA, a

chemically induced umor isolaed rom a AP1-knockou mouse, we observed he same

dichoomy in presened pepide reperoires beween AP-deficien and AP-proficien

cells (Fig 1B). MCA cells only presened he rh4 pepide, bu no he AP-dependen

conrol. Imporanly, resoraion o he mouse AP1 gene in his umor resuled in

decreased simulaion o he rh4-specific CLs and simulaneously in an increased

simulaion o he conrol CLs (Fig 1B). Tis indicaed ha MCA .AP1 cells display a

mixed pepide reperoire in heir MHC-I molecules comprised o boh AP-dependen

and -independen pepides. Te inroducion o he AP1 subuni did no ully resore

AP uncion in hese cells bu showed an inhibiory effec o AP on rh4 presenaion.

Tis effec was poeniaed by IFN-γ reamen o MCA.AP1 cells, a srong inducer o

he convenional anigen processing and presenaion machinery. IFN-γ-reaed MCA.

AP1 cells displayed he oher exreme o he pepide profile, in ha he rh4 pepide

was virually absen rom he cell surace (Fig 1B). IFN-γ reamen o parenal AP-

knockou MCA cells did no abrogae rh4 presenaion, suggesing ha deficiency o

AP is sufficien or he surace display o rh4/MHC-I complexes.

Nex, we sudied he presenaion o he rh4 pepide by dendriic cells, which are

highly efficien anigen presening cells (APCs). Inroducion o he viral evasion proein

UL49.5 ha arges AP uncion40 resuled in modes inhibiion o pepide ranspor

and MHC-I presenaion o hese cells as we previously showed41. Conrol CLs specific

or a AP-dependen epiope were sill capable o recognizing hese UL49.5-expressing

dendriic cells, albei o lower exen, atesing o he parial inhibiion o AP (Fig 1C).

Neverheless, his parial AP impedimen led o he emergence o he rh4 pepide on

hese dendriic cells (Fig 1C).

Tese daa corroboraed our previous work and showed ha he endogenous rh4

proein is ubiquiously presen in cells, bu is C-erminal epiope is only presened

by MHC-I on cells harboring a parially impaired AP uncion. Furhermore, hese

resuls demonsraed ha he pepide reperoire rom he convenional proeasome-AP

pahway can co-exis wih ha o alernaive roues and ha AP uncion mediaes he

shi beween hese reperoires.

Trh4 peptide presentation by primary tissue cells

rh4 presenaion was hus ar sudied wih in vitro culured cell lines. o obain a

broader profile o rh4 pepide presenaion by primary issues we examined a panel

o ex vivo isolaed issue cells rom organs o wild ype mice and AP1-knockou mice.

Parenchymal cell populaions were purified by mechanical and enzymaic disrupion

o organs ha were perused wih heparin. Ex vivo depleion o CD45+ hemaopoieic

cells, including anigen presening immune cells resuled in purified epihelial wih

sromal cell populaions. issue cells rom AP1-knockou mice were recognized by he

rh4-specific CLs, excep rom spleen and liver (Fig 2A). Ineresingly, he degree o

recogniion varied beween he organs and was paricularly high or bone marrow, lymph

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R M A

R M A - S

C 4 . 4 - 2 5

0

10

20

30A

B

Number of target cells

5 0 0 0

1 0 0 0 0

2 0 0 0 0

1 0 0

1 0 0 0

1 0 0 0 0

1 0 0 0 0 0

0

10

20

30

40

50

R M A

R M A - S

C 4 . 4 - 2 5

0.0

2.5

5.0

7.5

10.0

12.5

Trh4 CTL Control CTL

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

0

5

10

15

C D1.UL49.5D1.vector

MCA

MCA, IFNγ treated

MCA.TAP1, IFNγ treated

MCA.TAP1

I F N γ

r e l e a s e ( n

g / m l )

I F N γ

r e l e a s e ( n g / m l )

I F N γ


______ ________

1 0

1 0 0

1 0 0 0

1 0 0 0 0

0

25

50

75

100

0

5

10

15

0

Figure 1. Te rh4 peptide is only presented on cells with impaired AP function. Pepidepresenaion was evaluaed by incubaing CLs (rh4-specific or conrols) wih arge cells andmeasuring IFN-γ levels in he supernaan by ELISA aer culure or 18 hours. Te rh4-specific CLclone recognizes he MCLRMAVM pepide rom he rh4 proein; he conrol cells recognize heAP- and proeasome-dependen pepides (A) CCLCLVFL, an MuLV-derived pepide, and (B, C)he H-2D b-leader derived pepide AMAPRLLL. Te arge cells are (A) RMA, a AP-proficienlymphoma cell line; RMA-S, a TAP2-muan varian o RMA; and C4.4-25, a β2m-deficien lymphomacell line; (B) MCA fibrosarcoma cells derived rom AP1 -/- mice eiher unreaed or reconsiued

wih he TAP1 gene (MCA.AP1) and, where indicaed, pre-reaed wih IFN-γ o boos he anigenprocessing and presenaion machinery beore culure wih he CLs; (C) D1 dendriic cells ransduced

wih he viral AP-inhibior UL49.5 (D1.UL49.5) or an empy vecor (D1.vecor). Means and sandarddeviaions o riplicaes are shown rom one ou o hree independen experimens.

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nodes and hymus, suggesing ha he epihelial and connecive issue cells o heseorgans efficienly suppor MHC-I anigen presenaion. Parenchymal cell populaions

o liver and spleen appeared o lack rh4/MHC-I complexes and we speculae ha

hese organs do no express he processing enzymes or rh4 pepide liberaion.

None o he issue populaions o wild ype mice was recognized by he rh4-specificCLs (Fig 2A). Analysis o rh4 gene expression by he issues revealed no difference

beween wild ype and AP1-knockou cell populaions, indicaing ha he rh4 gene

was expressed by all issue cells o he wild ype mouse as well (Fig 2B). o deermine

rh4 gene expression in hymus in more deail, we perormed expression analysis

on separaed hymus subpopulaions ha are known o mediae negaive selecion42.rh4 ranscrips were deeced in medullary hymic epihelial cells (mEC), dendriic

cells and macrophages (Fig 2C). In addiion, rh4 was also expressed by hymocyes.

Noably, hymus sromal cells rom wild ype mice did no presen he rh4 epiope in

conras o heir AP1-knockou counerpars, suggesing ha negaive selecion o hisCL specificiy in wild ype mice is negligible.

Tese resuls implied ha rh4 is ubiquiously expressed in situ in he body, including

hymic cells ha mediae negaive selecion, bu ha he convenional AP-aciliaed

pepide reperoire prevens is MHC-I presenaion. As a consequence, cell recepors

wih rh4/H-2D b specificiy will no be deleed rom he CL reperoire in normal mice.

Trh4 is a stable and high affinity peptide for binding to H-2D b

In order o sudy he underlying mechanisms leading o he absence o he rh4 pepide

on cells wih normal anigen processing uncions we assessed he capaciy o he rh4pepide o compee or MHC-I loading as weak ineracions wih he presening molecule

H-2D b migh explain is ailure o simulae cells. I is known ha he ER-residen

pepide loading complex acively selecs high qualiy pepides or MHC-I binding11, 43 and, poenially, weak binding affiniy migh hus preclude rh4 loading. We deermined

he binding affiniy o rh4 o H-2D b as well as he dissociaion rae o hese complexes

and compared hese values wih hose or a viral CL epiope and five oher umor-

associaed CL epiopes (Fig 3). Te measured pepide binding affiniies showed ha

rh4 was posiioned in he group o high affiniy binders, comparable o he viral gp33

epiope rom LCMV (Fig 3A). rh4 was clearly disinguishable rom low affiniy pepideslike he MDM2- and gp100-derived epiopes. Moreover, he sabiliy o rh4/H-2D b

complexes, a parameer ha srongly associaes wih immunogeniciy 44 , was superior o

all he oher esed pepides (Fig 3B). Aer 6 hours, virually all rh4/H-2D b complexes

were sill deeced, whereas he umor-associaed pepides showed a as decay.Considering hese resuls, we concluded ha he rh4 pepide binds o is presening

MHC-I molecule wih high affiniy and sabiliy. Tese eaures would raher aciliae

han avoid is presenaion by normal cells.

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Bone Marrow Lymph Nodes Thymus

Liver

I F N γ

r e l e a s

e ( p g / m l )

Lung Spleen

TAP1-/-

0

2 5 0 0 0

5 0 0 0 0

7 5 0 0 0

1 0 0 0 0 0 0

2 5 0 0 0

5 0 0 0 0

7 5 0 0 0

1 0 0 0 0 0 0

2 5 0 0 0

5 0 0 0 0

7 5 0 0 0

1 0 0 0 0 0

C57BL/6

Number of target cells

0

2

4

6

8

10

12

R e l a t i v e m R N A e x p r e s s

i o n

L u n g

L i v e r

S p l e e n

T h y m u s

L y m

p h N o d e s

B o n e M a r r o w

B r a i n

R e l a t i v e m R N A e x p r e s s i o n

m T E C

C D 8 0 h i g h

T h y m o c y t e s

M a

c r o p h a g e s

D C

m T E C

C D 8 0 l o w

TAP1-/-

C57BL/6

A

B C

0.0

0.5

1.0

1.5

2.0

216

30

0

250

500

750

1000

5000

15000

25000

0

250

500

750

1000

5000

15000

25000

I F N γ

r e l e a s e ( p g / m l )

Figure 2. rh4 is widely expressed and presented on AP-deficient primary tissue cells. (A) Teindicaed organs/issues were colleced rom C57BL/6 and AP1-/- mice and processed in vitro bymechanical and enzymaic disrupion. CD45+ cells were depleed rom he isolaed issue cells by use omagneic CD45 MicroBeads and he remaining cells were incubaed wih he rh4-specific CD8+ -cellclone and IFN-γ producion deermined. (B-C) oal RNA was exraced rom he isolaed primary cellsderived rom (B) he indicaed issues/organs o C57BL/6 and AP1 -/- mice or (C) he specific hymic sub-populaions o C57BL/6 mice. cDNA was synhesized rom he RNA samples and Trh4 mRNA expressiondeermined by quaniaive PCR wih normalizaion o he (B) GAPDH or (C) β-actin housekeeping

genes and expressed relaive o (B) hymus AP1-/-

and (C) mEC CD80high

Trh4 mRNA levels. Meansand sandard deviaions o riplicaes are shown rom one ou o hree independen experimens.

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MHC availability is not the limiting step for Trh4 loading and

presentation

Nex, we esed wheher he availabiliy o MHC-I molecules was limiing or he

presenaion o rh4. Higher expression levels or he heavy chain migh enable he rh4

pepide o be loaded ino H-2D b molecules even in he presence o he pepide reperoire o

he convenional processing pahway. A reroviral expression vecor conaining he H-2D b

gene was inroduced ino AP-posiive RMA cells and MHC-I proein levels were analysed by flow cyomery. Te surace display o H-2D b molecules increased nearly wo-old,

indicaing ha exra availabiliy o heavy chain proeins indeed enhances pepide loading

and surace presenaion o pepide/MHC-I complexes (Fig 4A). Neverheless, analysis

o rh4 presenaion showed ha addiional heavy chains did no bring his pepide o

he cell surace (Fig 4B). Tese daa suggesed ha he supplemenary MHC-I moleculespredominanly accommodaed pepides rom he classical reperoire. Furhermore, a hree-

old increase o H-2D b molecules a cell surace o AP-deficien RMA-S cells neiher

enhanced he surace display o rh4 pepides (Fig 4A and 4B). Tis resul suggesed ha

AP deficiency, which blocks he presenaion o he classical pepide reperoire, creaes

he opporuniy or all available rh4 pepides o be loaded, irrespecive o MHC-I levels.ogeher, hese resuls showed ha he availabiliy o MHC-I molecules is no he rae-

limiing sep or he resriced presenaion o rh4 by processing-deficien cells.

0 1 2 3 4 5 6 7

0

20

40

60

80

100

Time (h)

A Binding Stability

P e p t i d e / D b b

i n d i n g

i n d e x

B

0

1

2

3

4

5

6

7

8

10-510-410-310-210-1 100 101 102 103

% r

e m a i n i n g p e p t

i d e / D b

Peptide concentration (µM)

Trh4379-387

gp3333-42

CEA571-579

MDM2441-449WT1

126-134

cyc20-28

gp10025-33

Figure 3. Binding and stability of rh4 peptide/H-2Db complexes. (A) Te binding affiniy o herh4 pepide o H-2D b was evaluaed. RMA-S cells were incubaed wih varying concenraions o heindicaed H-2D b binding pepides and H-2D b cell-surace levels aer 4h incubaion were deermined byflow cyomery. Te binding index o pepides o MHC was calculaed as he raio o he mean fluorescenceinensiy (MFI) beween loaded and unloaded cells. Te index o a conrol H-2 b binding pepide wasone in all concenraions (no depiced). (B) Te decay o he rh4/H-2D b complexes was esed byincubaion o RMA-S cells wih he indicaed pepides and evaluaion o surace H-2D b levels by flowcyomery over ime. Te daa are he percenages o remaining pepide/H-2D b complexes on he cellsurace as compared o iniial levels. Resuls shown are represenaive o hree independen experimens.

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o he anigen, a successul compeiion wih he pepide reperoire o he convenional

pahway was achieved. o assess he expression level o rh4, quaniaive PCR analysis

was perormed, since anibodies agains he proein were no available. Te rh4 gene in

our RMA .rh4 cells was expressed 120 imes higher han he endogenous levels in RMA

cells (Fig S1). Te expression o he shor rh4 ranscrip did no vary beween he cell

lines, because his cDNA was no involved in gene ranser (Fig S1). We anicipaed ha

over-expressed rh4 proein was sill ER localized like he endogenous proein, since

a previous sudy confirmed his using HA-agged rh435. Moreover, he over-expressed

rh4 is no shutled o he cyosol or proeasome-mediaed degradaion, because

epoxomicin did no inhibi he presenaion o he rh4 pepide by hese RMA.rh4

cells (Fig S2). Ineresingly, he proeasome inhibior raher increased he presenaion

o he AP-independen epiope, again confirming ha he classical processing roue o

proeasome-AP hampers he presenaion o pepides rom alernaive pahways. We hen examined he quaniy range ha was needed or he presenaion o rh4 on

AP-posiive cells. RMA.rh4 cells were sored in hree populaions on basis o fluorescen

GFP proeins levels, which corresponded o rh4 levels due o a coupled ranslaion

iniiaion by an inernal ribosome enry sie in our gene consruc. Gene expression analysis

showed ha he hree RMA.rh4 populaions harbored approximaely 800, 400 and 20

imes he amoun o endogenous rh4, respecively (Fig 5B). Again, over-expression o

he long rh4 ranscrip did no influence he expression o he shorer isoorm. RMA.

rh4 cells wih 20 imes over-expression were recognized by he CL clone, bu jus

above he quaniy hreshold (Fig. 5C). RMA.rh4 cells wih 400 imes over-expressionpresened he epiope o convincing levels. However, his high expression level was sill

no sauraing, as RMA.rh4 cells wih 800 imes over-expression were clearly beter

recognized by he rh4-specific CL clone (Fig 5C). Te ac ha hese exreme levels o

rh4 expression were needed o reach opimal pepide presenaion in AP-posiive cells

implied ha he convenional pepide reperoire enering he ER via AP mediaes srong

resisance o he presenaion o he alernaively processed rh4 pepide. Imporanly,

vigorous over-expression o his one ranscrip did no aler or inhibi he presenaion

o he AP-mediaed conrol pepide (Fig 5C). We wondered i he over-expressed Trh4

gene in RMA.rh4 cells would reach ranscrip levels comparable o hose o classicalanigens. Te endogenously expressed reroviral gag gene encodes he immunodominan

H-2D b-presened epiope CCLCLVFL, which is AP- and proeasome-dependen45.

Whereas endogenous rh4 expression in he umor cells was very low, he over-expressed

gene approached levels comparable o he gag gene (Fig. S3). Tis indicaed ha he rh4

epiope is no presened by AP-posiive cells because is expression level is oo low.

Increased rh4 expression in proessional anigen-presening cells (dendriic D1 cells)

also induced recogniion by rh4-CLs (Fig S4). Simulaion o he convenional

processing roue hrough LPS-mediaed mauraion o hese cells decreased rh4 pepide

presenaion, supporing our noion on compeing pepide reperoires (Fig. S4).

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A

C Trh4-CTL Control CTL

M e d i u m

R M A

R M A . T r h 4 h i g h

R M A . T r h 4 i n t e r m .

R M A . T r h 4 l o w

M e d i u m

R M A

R M A . T r h 4 h i g h



Trh4 Trh4 splice variantB

R M A

R M A . L Z R S

R M A . T r h 4

R M A - S

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Figure 5. rh4 peptide is presented on AP-proficient cells upon over-expression of rh4. (A)

Reaciviy o rh4-specific -cell clone agains AP-proficien RMA cells and AP2-deficien RMA-Scells ransduced or no wih he Trh4 gene (RMA.rh4, RMA-S.rh4) or an empy vecor (RMA.LZRS) as deermined by IFN-γ producion. Four old serial diluions o arges were perormed saringa 20,000 arge cells as indicaed by he grey scale bars. (B) mRNA expression, in he indicaed cells,o Trh4 and a naural Trh4 splice varian as deermined by quaniaive PCR using specific primers odisinguish boh ranscrips. Te splice varian, used as a conrol, is a shorer ranscrip ha does noconain he sequence o he rh4 pepide MCLRMAVM. Trh4 expression levels were normalized wihhe β2m housekeeping gene and expressed relaive o he RMA sample. (C) RMA cells over-expressingrh4 proein (RMA.rh4) eiher weakly or a medium or high levels (low, inerm. or high respecively)

were incubaed wih rh4-specific or MuLV conrol cell clones and IFN-γ producion measured. woold (conrol CLs) or our old (rh4-CLs) serial diluions o arges were perormed saring a20,000 arge cells and are indicaed by he grey scale bars. Means and sandard deviaions o riplicaes

are shown rom one ou o hree independen experimens.

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We concluded ha he AP-independen pepide reperoire needs o compee wih

he convenional reperoire or loading and presenaion by MHC-I. Tis resisance o

he alernaive reperoire is swily alleviaed by decreasing levels o he AP ransporer.

Te newly emerging pepides on AP-impaired cells hereore represen immunogenic

neo-anigens and consiue unique CL arges or AP-deficien umors and cells

ineced wih immune evading herpes viruses.

| DISCUSSION

MHC-I presened pepides consiue proein breakdown inermediaes ha are rescued

rom complee desrucion in he caabolic milieu o he cyosol. Te oal pool o cyosolic

pepides ormed by proeolysis is very diverse and esimaed o be composed by millions o

pepides2, 3, 46-48. From his pool only a small reperoire is seleced or presenaion by MHC-I

molecules a he surace o cells. In our curren sudy we demonsrae ha his reperoireselecion is largely governed by he pepide ransporer AP, which pumps pepides rom

he cyosol ino he ER. Te AP complex behaved like a lever o conrol o shi he

presened reperoire gradually owards AP-independen or AP-dependen pepides.

Cells wih normal anigen processing uncion did no presen he AP-independen

rh4-derived pepide a he cell surace, despie he ac ha his pepide is generaed in

AP-posiive cells and is capable o orming sable complexes wih H-2D b (Fig 3). Modes

inhibiion o AP uncion, however, resuled in surace display o his rh4/D b complex.

In addiion, parial deficiencies sill allowed he presenaion o AP-dependen pepide

species, yielding a mixed pepide reperoire o boh pools. Te highes presenaion o rh4pepide occurred when AP uncion was compleely blocked. Tese daa revealed ha AP

suppors a highly compeiive environmen or class I loading in he ER mos likely due o

he overwhelming flow o pepide species ha i pumps rom he cyosol. Parial inhibiion o

AP uncion alleviaes his compeiion and induces he presenaion o novel pepides and

gradually inhibis he presenaion o he classical AP-dependen pool. Tis hypohesis is

corroboraed by he finding ha over-expression o he rh4 proein resuls in higher amoun

o pepide epiopes ha are able o successully compee or presenaion in processing-inac

cells (Fig 5). Alernaively, he selecive rh4 presenaion could be explained by difference

in organizaion o he pepide-loading complexes in he absence o AP. A change insoichiomery o he pepide-loading complex migh avor an alernaive pepide reperoire.

Tis alernaive hypohesis is, however, less likely because over-expression o he rh4 proein

is no expeced o change he loading complex, while i sill leads o surace presenaion

o he CL epiope by H-2D b. Tis orced presenaion o a AP-independen pepide was

operable in leukemia cells as well as in primary dendriic cells (Fig 5 and Fig S4), illusraing

he general characer o his mechanism. We hereore conclude ha AP aciviy normally

conribues o he reenion o his AP-independen pepide inside he cell. Apparenly, no

all pepides ha can be presened on basis o accessibiliy o he ER and o binding affiniy

are acually presened. A consequence o his reasoning is ha pepide availabiliy is no he

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rae limiing sep in anigen presenaion. Te ac ha MHC-I surace expression can be

enhanced by gene ranser o heavy chains, urher suppors his noion (Fig. 4).

Te sudied rh4 pepide represens a much broader reperoire o AP-independen

pepides, as we have shown ha AP-deficien cells are recognized by a diverse pool o

CL clones wih disinc MHC resricion paterns36, 41. Ineresingly, he non-classical

MHC molecule Qa-1 b seems o play a dominan role in he presenaion o hese AP-

independen anigens49-51. Recenly, we showed he exisence o he human equivalen

o his novel CL caegory 52, 53. Tese pepide sequences, called EIPP (-cell epiopes

associaed wih impaired pepide processing) are no presened by normal cells and we

can speculae, based on he resuls o he presen sudy, ha one mechanism governing he

absence rom normal cells is relaed o he low expression level o he cognae proeins. In a

parallel sudy, a proeasome- and AP-independen umor anigen rom he signal sequence

o he preprocalcionin proein (ppC16-25

) was ound o represen a human EIPP, in

ha his HLA-A2 presened pepide was selecively presened by umor cells wih AP-

deficiency 54. Tis pepide is liberaed in he ER lumen by sequenial cleavage wih SP and

SPP56 and is a clear example o he alernaive AP-independen pepide reperoire.

We like o emphasize ha no all AP-independen pepides ail o be presened

by processing inac cells. Mass-specromery analyses have revealed ha par o his

reperoire can be deeced on he surace o AP-proficien cells25-27. We anicipae ha

he expression levels o hese proeins or he rae o pepide ormaion is higher han ha

o EIPPs. Consequenly, he immunogeniciy o hese pepides is expeced o be much

lower, due o cenral and peripheral cell olerance, as hese pepides are derived rom

ubiquiously expressed housekeeping proeins.

Te emerging picure o MHC-I anigen processing incorporaes novel proeolyic

enzymes nex o he mulicaalyic proeasome complex as he cenral player 6-9. Mos o

hese novel breakdown sysems liberae pepides in he cyosol and produce subsraes

ha sill need AP ranspor or MHC-I loading. However, processing by ye oher

proeolyic sysems deliver pepides ha are presened in a AP-independen way. Te

above menioned SP and SPP proeases produce such AP-independen pepides wihin

he ER and proproein converases like PC7 and urin have been shown o aciliae

AP-independen presenaion in he secreory roue29-32. Ineresingly, our preliminary

daa show ha presenaion o he rh4 pepide is independen o hese known enzyme

sysems, indicaing ha ye oher pahways exis.

In his sudy we show ha AP influences he balance o pepide reperoires and ha

poenial MHC-I ligands compee or loading and surace display. AP down-modulaion

aciliaes he presenaion o he suppressed reperoire. Imporanly, inhibiion o his

processing botleneck is a common eaure in cancer55, 57 and AP is also a requenly

argeed molecule by mos viruses ha cause lielong inecions15. Te direc consequences

are a generalized decrease in MHC-I presenaion and he emergence o alernaive

pepide reperoire. Tese AP-independen pepides migh consiue an imporan line

o hos deense ha migh be exploied in herapeuic inervenion sraegies.

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MATERIAL AN D METHODS

Cell lines and mice

Te umor cell lines RMA, RMA-S (AP2 deficien), C4.4-25 (β2m deficien), MCA

(AP1 deficien) and he D1 dendriic cell line have been described previously 36, 41, 49.Cell varians overexpressing H-2D b or rh4 (accession number UniProB/Swiss-Pro

Q9D69 LASS5_MOUSE) were generaed by reroviral gene ranser using he LZRS

vecor conaining GFP behind an inernal ribosome enry sie50. RMA.rh4 cells were

differenially sored according o GFP levels o generae varians wih low, inermediae

and high expression o rh4. MCA.AP1 is a varian o MCA fibrosarcoma where he

mouse AP1 gene was inroduced. UL49.5 is a gene rom he Bovine Herpes Virus-1 and

blocks mouse AP aciviy 40, 50. Generaion and culure o cell clones was described

previously 36, 45, 50. CD8+ cell clones used in his sudy: Qa-1-resiced CL B12i is specific

or he AP-dependen pepide Qdm (AMAPRLLL); H-2D b-resriced CL clone 1 isspecific or he AP-dependen MuLV gag-leader pepide (CCLCLVFL); H-2D b-resriced

CL clone B5 is specific or he AP-independen rh4 pepide (MCLRMAVM). All

cells were culured in complee IMDM medium (Invirogen, Carlsbad, CA) conaining 8%

hea-inacivaed FCS, 100 U/mL penicillin, 100 μg/mL srepomycin (Lie echnologies,

Rockville, MD), 2mM L-gluamine (Invirogen) and 30 μmol/L o 2-mercapoehanol

(Merck, NJ, USA) a 37°C in humidified air wih 5% CO2.

C57BL/6 mice were purchased rom Charles River Laboraories (France and

Germany) or were bred a he German Cancer Research Cener (DFZ) and housed in

he animal aciliy o he Leiden Universiy Medical Cener and DFZ under specifiedpahogen-ree condiions. Mice were used beween 8 and 12 weeks o age. AP1-

knockou mice were purchased rom Jackson laboraories. Experimens were perormed

in accordance wih naional legislaions and insiuional guidelines and were approved

by he local ehical commitees.

T-cell activation assays and flow cytometry

cell aciviy was measured by IFN-γ secreion. Supernaans were harvesed aer

overnigh incubaion o cells wih arge cells and concenraions o IFN-γ were

measured by ELISA as previously described58

. Surace expression o H-2D b

molecules was deermined using purified mouse ani-H-2D b mAb (clone 28.14.8S; BD Biosciences,

NJ, USA) ollowed by allophycocyanin labeled goa ani-mouse Ig (Souhern Bioech,

Birminghan, USA). Cells were analyzed using a FACS Calibur wih Cellques soware

(BD Biosciences) or Flowjo soware (ree Sar, Ashland, OR).

Quantitative PCR analysis

oal RNA isolaion and cDNA preparaion was perormed using RNeasy Mini i

(Qiagen, Maryland, USA). 500 ng o purified oal RNA was used o synhesize cDNA

using High Capaciy RNA-o-cDNA i (Applied Biosysems, Foser Ciy, USA).

Quaniaive PCR on shor and long ranscrips o rh4 was done as described beore 36.

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18/25

Lung issue ragmens were colleced o genleMACS C ubes (Milenyi Bioec, BergischGladbach, Germany) and mixed wih 0.05mg/mL DNase I and 250 U/mL collagenase

IV dilued in serum-ree medium (5 mL enzyme mix/2 lungs). ubes were ranserred

o genleMACS Dissociaor (Milenyi Bioec) and he proocol was run according o

manuacurer’s recommendaions. Cell suspension was passed hrough a cell srainero remove cell clumps. Livers were colleced o genleMACS C ubes and mixed wih

0.05 mg/mL DNase I and 500 U/mL collagenase IV dilued in serum-ree medium (5 mL

enzyme mix/2 livers). Te ragmens were incubaed a 37 °C or 30 minues under slow

mixing. Aer incubaion, ubes were ranserred o genleMACS Dissociaor and he

proocol was run according o he manuacurer’s recommendaions. Cell clumps wereremoved wih a cell srainer.

Blood-derived CD45+ cells were depleed rom he issue cell suspensions by

negaive selecion wih magneic CD45 MicroBeads (Milenyi Bioec) according o

manuacurer’s proocol. Conaminaion wih CD45 cells was esimaed by saining wihanibodies agains he epihelial cell marker CD326 (Ep-CAM) and CD45.

For he isolaion o hymic subpopulaions, hymic lobes were cleaned o a and

connecive issue, cu in litle pieces and sirred or 10 min in 15mL RPMI medium a

room emperaure o release hymocyes. issue ragmens were hen resuspended in

1 mL medium per hymus conaining 0.2 mg/mL Collagenase IV (Worhingon), 10mM

HEPES and 2% FCS. Te mixure was slowly sirred or 15 min a 30°C, released cellsremoved and resh enzyme mixure added or a oal o 3 incubaions. Te remaining

ragmens were digesed wih a mixure o Collagenase IV and Neural Proease

(0.2 mg/mL each; Worhingon), 25 mg/mL DNaseI (Roche), 10 mM HEPES and 2%FCS in RPMI1640. Five incubaions or 25 min a 37°C were perormed. For DC and

macrophage isolaion all cells rom he collagenase incubaions and he firs round ocollagenase/neural proease digesion were pooled, washed and rosetes dissociaed by

5 min incubaion a 37°C in PBS conaining 25mM EDA. Cells were sained wih ani-

CD11c microbeads (Milenyi Bioec), run on an AuoMACS (Milenyi Bioec) using

he “Possel_S” program, blocked wih ani-FcR mAb 2.4G2 supernaan including 5 %

ra serum and sained wih ani-CD11c-PE (clone HL3; BD), ani-F4/80-FIC (cloneCI:A3-1; Seroec) and PI. Tymic epihelial cells were enriched by pooling cells rom

collagenase/neural proease digesion rounds wo o five, ollowed by saining wihani-CD45 microbeads (Milenyi Bioec), run on an AuoMACS using he “Deplee”

program, blocked wih ani-FcR mAb 2.4G2 supernaan and sained wih ani-Ly51-FIC (clone 6C3; BD), ani-CD80-PE (clone 16-10A1; BD), ani-EpCAM-Alexa 647

(clone G8.8; BD), ani-CD45-PECy5 (clone 30-F11; BD) and PI. Dendriic cells were

idenified as CD11c+F4/80- and macrophages as CD11c-/lo and F4/80+. mECs were

idenified as CD45-Ly5.1-EpCAM+ and sored according o heir CD80 expression, as

CD80hi or CD80lo, represening he op and botom 30% o he populaion. Cell soring was perormed wih a FACSAriaI cell sorer (Becon Dickinson).

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ACKNOWLEDGEMENTS

Financial suppor was received rom Poruguese Foundaion For Science and echnology

(MCES) Porugal (SFRH/BD/33539/2008 o CCO), he AICR (09-776 o MS) and he

Duch Cancer Sociey (UL 2007-3897 o BQ). Te auhors would like o acknowledge MargiH. Lampen, Ursula J. E. Seidel and Pro. Dr. C. Melie or criical reading o he manuscrip.

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Alternative Antigen Processing and Presentation Pathways by Tumors - 02

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