Tumor Biology and Immunology

ERAP1-Dependent Antigen Cross-PresentationDetermines Efficacy of Adoptive T-cell Therapyin MiceKarin Schmidt1, Christin Keller1, Anja A. K€uhl2, Ana Textor3, Ulrike Seifert1,Thomas Blankenstein3,4,5, Gerald Willimsky4,6, and Peter-Michael Kloetzel1,5

Abstract

Cytotoxic T lymphocytes can reject established tumors if theirtarget peptide is efficiently presented by MHC class I molecules(pMHC-I) on the surface of cancerous cells. Therapeutic successupon adoptive T-cell transfer (ATT), however, requires additionalcross-presentation of the same pMHC-I on noncancerous cells.Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzymethat customizes the N-terminus of proteasome-generated pep-tides so they can be loaded onto MHC-I molecules in the endo-plasmic reticulum (ER). We show here that ERAP1 is criticallyinvolved in the process of tumor rejection and assumes a dual roleby independently operating on both sides. Direct presentation oftwo MHC-I–restricted epitopes of a cancer-driving transplanta-tion rejection antigen through ERAP1 moderately affected tumorrejection by adoptively transferred T-cell receptor gene–modified

T cells in each case. ERAP1 expression by antigen cross-presentingcells of the ATT recipients was critical for expansion of therapeuticmonospecific T cells and correlated with tumor rejection. Specif-ically, lack of ERAP1 expression in the ATT recipient's noncan-cerous cells enabled progression of pMHC-I–positive, IFNg-responsive tumors, despite the presence of antigen-specific func-tional cytotoxic T lymphocytes. These data reveal a decisive rolefor ERAP1 in T-cell–mediated tumor rejection and will enhancethe choice of MHC-I–restricted epitopes targeted by adoptive T-cell transfer.

Significance: This study demonstrates a role of ERAP1 in theefficacy of adoptive T-cell transfer and has potential to improvepersonalized T-cell therapy for solid tumors. Cancer Res; 78(12);3243–54. �2018 AACR.

IntroductionOne of the most effective therapies for patients with cancer is

the transfer of tumor-infiltrating T lymphocytes with objectiveresponse rates >50% being achievable (1). Successful elimina-tion of cancer depends on both, efficient direct presentationof the targeted MHC-I epitope, and on the cross-presentationof the same MHC-I epitope on noncancerous cells (2–5).

Moreover, adequate T-cell persistence decides on the effective-ness of adoptive T-cell transfer (ATT), because the number oftransferred T cells and the degree of their persistence in periph-eral blood correlates with cancer regression (1, 6).

On the side of the target epitope, classical processing of MHC-Iligands involves three consecutive steps assumed by proteasomes,the transporter associated with antigen presentation (TAP), andendoplasmic reticulum aminopeptidase 1 (ERAP1). The majorityof peptides presented on MHC-I molecules on the cell surface isgenerated by proteasomes and possesses a C-terminal residuesuitable to act as anchor for MHC-I binding (7). Those peptidesare transported into the endoplasmic reticulum (ER) by TAP (8). Inmany cases the epitope-containing peptides are N-terminallyelongated, requiring further optimization in the ER. Here, ERAP1trims amino acid residues that flank the N-termini of antigenicprecursors (9, 10). In the context of cancer immunotherapy, itwas shown in mice that inhibition of ERAP1 caused increasedtumor immunogenicity through direct presentation of a neo-epitope targetable by specific T cells (11). But when tumors evadedtherapy with T-cell receptor (TCR) gene-modified T cells, recog-nition of IFNg-resistant cancer variants by cytotoxic T lymphocytes(CTL) was reconstructible by overexpression of ERAP1 in vitro (12).

In contrast with the well-analyzed direct presentation ofantigens, the procedures of antigen cross-presentation remainenigmatic. Considering the necessity of N-terminal trimmingof peptides, Saveanu and colleagues (13) demonstrated thatinsulin-responsive aminopeptidase is required for efficient cross-presentation of endocytosed ovalbumin (OVA) and of phago-cytosed antigen. Information on the role of ERAP1 in antigencross-presentation is barely available and is mainly restrictedto the analysis of the model antigen OVA (13–15). Here,

1Institute of Biochemistry, Charit�e—Universit€atsmedizin Berlin, corporatememberof Freie Universit€at Berlin, Humboldt-Universit€at zu Berlin, and Berlin Institute ofHealth (BIH), Berlin, Germany. 2iPath.Berlin—Immunopathology for ExperimentalModels, Charit�e—Universit€atsmedizin Berlin, corporate member of Freie Uni-versit€at Berlin, Humboldt-Universit€at zu Berlin, and Berlin Institute of Health(BIH), Berlin, Germany. 3Max-Delbr€uck-Center for Molecular Medicine, Berlin,Germany. 4Institute of Immunology, Charit�e - Universit€atsmedizin Berlin,corporate member of Freie Universit€at Berlin, Humboldt-Universit€at zu Berlin,and Berlin Institute of Health (BIH), Berlin, Germany. 5Berlin Institute of Health,Berlin, Germany. 6German Cancer Research Center (DKFZ), Heidelberg, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

G. Willimsky and P.-M. Kloetzel contributed equally to this article.

Current address for U. Seifert: Friedrich Loeffler Institute for Medical Microbi-ology, University Medicine Greifswald, Greifswald 17475, Germany.

Corresponding Authors: Karin Schmidt, Institute of Biochemistry, Charit�eplatz1, 10117 Berlin, Germany. Phone: 4930-4505-28395; Fax: 4930-4505-28921;E-mail: k.schmidt@charite.de; and Peter-Michael Kloetzel, Institute of Bio-chemistry, Charit�eplatz 1, Berlin 10117, Germany. Phone: 4930-4505-28071;Fax: 4930-4505-28921; E-mail: p-m.kloetzel@charite.de

doi: 10.1158/0008-5472.CAN-17-1946

�2018 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 3243

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

we demonstrate the decisive role of ERAP1 for therapeuticoutcome, showing that for optimal efficacy of ATT, ERAP1must operate on both sides, direct antigen presentation incancerous cells and antigen cross-presentation in the ATTrecipient's cells.

Materials and MethodsMice

LoxP-Tag and LoxP-Tag � Alb-Cre mice were describedpreviously (16, 17). Erap1�/� mice were provided by K. Rock(18). Erap1�/� mice were crossed to LoxP-Tag and Alb-Cremice to obtain Erap1 � LoxP-Tag � Alb-Cre mice. SCID mice(C.B-17, strain code 236) were purchased from Charles RiverLaboratories. Rag�/� mice (B6.129S6-Rag2tm1Fwa), Rag�/� �gc�/� mice (B10;B6-Rag2tm1FwaIl2rgtm1Wjl), and CD45.1 micewere purchased from Taconic and were bred in our animalfacilities at the FEM Charité-Universitätsmedizin Berlin.Erap1�/� mice were crossed to Rag�/� mice to obtain Erap1�/�

� Rag�/� mice. P14 � Rag�/� mice were described previously(12). C57BL/6 mice were provided by the FEM at Charit�eUniversit€atsmedizin Berlin. Male or female mice ages 2 to9 months were used in animal experiments. All mouse studieswere approved by the Landesamt f€ur Gesundheit und Soziales,Berlin, Germany.

Hepatocellular carcinoma cell linesTo generate WT (Erap1þ/þ) TAgþ hepatocellular carcinoma

(HCC) and Erap1�/� TAgþ HCC lines, livers were removedfrom tumor-bearing Erap1 � LoxP-Tag � Alb-Cre mice.Tumor tissue was cut into pieces with scalpels and wasdigested in RPMI supplemented with 10% heat-inactivatedFCS (Biochrom), 1� penicillin/streptomycin (Biochrom),1 mg/mL collagenase (Sigma), and 1� trypsin (Biochrom)for 4 hours at 37�C in a humidified 5% CO2 incubator. Toprepare a single-cell suspension, digested tumor tissue wasfiltered through a 45-mm cell strainer (BD Biosciences) andwashed with PBS twice. Single-cell suspensions were culturedin RPMI supplemented with 10% heat-inactivated FCS, 1�penicillin/streptomycin, 2 mmol/L glutamine (Biochrom),and 50 mmol/L b-mercaptoethanol (AppliChem). AdherentHCC cells were detached from cell culture flasks with trypsin(Biochrom) and were frozen in 90% culture medium sup-plemented with 10% dimethyl sulfoxide. Cells were culti-vated for a maximum of 10 passages after thawing. HCC celllines were regularly authenticated by Western blot analysis,but were not tested for Mycoplasma contamination.

Tumor challenge and adoptive T-cell transferAge- and sex-matched mice were subcutaneously injected

into the right flank with 1 � 106 HCC cells in 200 mL PBS.Tumor size was measured with a caliper and the average tumorvolume was determined from the measurements along threeorthogonal axes (x, y, z). Tumor volumes were calculatedaccording to the formula V (mm3) ¼ (x y z)/2. On the day oftreatment mice received intravenous injections of either 1� 106

polyclonal CD8þ T cells, 5 � 104 gene-modified TCR-I T cells,or 5� 104 gene-modified TCR-IV T cells re-suspended in 200 mLPBS. A small amount of blood was taken from the facial vein ofthe mice one and/or 4 weeks after adoptive transfer. Animalswere sacrificed when the tumors reached 15-mm mean diam-

eter and animals were excluded from analysis if they died fromreasons unrelated to tumor burden. The experimenter was notblinded for the treatment groups.

Quantification and statistical analysisComparison of two groups was done by the Mann–Whitney

test. Comparison of more �3 groups was done by the Kruskal–Wallis test. Two-way ANOVA was used followed by Bonferronipost-test for multiple comparisons. All statistical analysis wasdone with GraphPad Prism software version 5.0 and consideredsignificant at �, P � 0.05; ��, P � 0.01; and ���, P � 0.001.

ResultsTwo MHC-I epitopes differ in their dependence on ERAP1-mediated N-terminal peptide trimming

The cancer-driving antigen SV40 T Antigen (TAg) contains twocodominant MHC-I–restricted epitopes, the H2-Db-restricted10mer TAg206–215 (SAINNYAQKL, TAg-I), and theH2-Kb

–restrict-ed 8mer TAg404–411 (VVYDFLKC, TAg-IV; ref. 19). In Textor andcolleagues (12), we showed that both standard and immunopro-teasomes generated TAg-I along with the potential epitope pre-cursors TAg205–215 (VSAINNYAQKL, 11mer) and TAg204–215(RVSAINNYAQKL, 12mer). When peptide translocation wasassessed, ATP-dependent transport was observed for TAg-I andthe 11mer precursor peptide. The 12mer TAg-I precursor peptidedid not translocate in an ATP-dependent fashion (Fig. 1A). TAP-dependent transport was confirmed by reduced accumulation ofATP signal in the presence of a high-affinity competitor peptide(Supplementary Fig. S1A). Upon analysis ofN-terminal trimmingof the precursor peptides by recombinant mouse ERAP1(rmERAP1), TAg-I was generated from the 12mer through the11mer intermediate (Supplementary Fig. S1B; Fig. 1B and C).Hence, though TAg-I is generated by rmERAP1, the consecutiveaction of proteasomes and TAP may even provide sufficientamounts of the 10mer.

In contrast, TAg-IV was difficult to detect after proteasomalcleavage, whereas the corresponding N-terminally extended epi-tope precursor peptides TAg403–411 (SVVYDFLKC, 9mer) andTAg402–411 (DSVVYDFLKC, 10mer) were predominantly pro-duced (12). In peptide translocation experiments, all threeTAg-IV–containing proteasomal cleavage products were trans-ported in dependence of ATP and TAP (Fig. 1D; SupplementaryFig. S1C). For the intermediate 9mer peptide precursor, translo-cation efficiency did not exceed the experimental threshold setby a control peptide (E5) not binding to TAP. When the TAg-IV–containing N-terminal precursor peptides were subjected tormERAP1-digestion, TAg-IV was generated from the 10mer pre-cursor peptide through the 9mer intermediate, and thus mayconstitute an ERAP1-dependent epitope (Fig. 1E and F).

Generation of primary wild-type and Erap1�/� TAgþ

hepatocellular carcinoma with IFNg-inducible antigen-processing machinery

Although both immunoproteasomes and TAP control thequantity of peptides being available for MHC-I loading, ERAP1regulates the quality of the presented peptides (20). To assessthe role of ERAP1 in vivo, TAgþ tumors genetically depleted forErap1�/� were generated through crossing of Erap1�/� mice toLoxP-Tag � Alb-Cre mice, which express TAg specifically inhepatocytes and develop TAgþ HCC and cholangiolar carcinoma

Schmidt et al.

Cancer Res; 78(12) June 15, 2018 Cancer Research3244

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

by the age of 3 to4months (Fig. 2A; refs. 17, 18).Erap1þ/þ� LoxP-Tag � Alb-Cre double-transgenic offspring and Erap1�/� �LoxP-Tag � Alb-Cre triple-transgenic offspring likewisedeveloped TAgþ HCC after about 3 to 4 months. Histologicalanalysis of the primary HCC revealed a similar appearance ofWT (Erap1þ/þ) TAgþ HCC and Erap1�/� TAgþ HCC (Fig. 2B).WT and Erap1�/� TAgþ HCC cell lines were subsequentlyestablished from primary HCC. Two cell lines of each WTand Erap1�/� TAgþ HCC were further characterized (referredto as HCC pair 1 and HCC pair 2). Expression or lack of ex-pression of ERAP1 was confirmed by Western blot (Fig. 2C).Furthermore, IFNg-signaling was examined having a focus onthe expression of components of the antigen-processingmachinery (APM). WT and Erap1�/� TAgþ HCC constitutivelyexpressed JAK1 and downregulated JAK2 within 24 hours afterstimulation with rmIFNg . A highly elevated expression ofSTAT1 and phosphorylation of STAT1 was noticed in WT andErap1�/� TAgþ HCC. Likewise, all HCC lines upregulatedexpression of APM components such as TAP1 and TAP2, andthe immunoproteasome subunits b1i, b5i, and b2i (Fig. 2C).Notably, WT HCC of pair 2 showed a higher basal expression ofERAP1, LMP7, and MECL1. This correlated with higheramounts of MHC-I in the absence of IFNg in this HCC line

(Fig. 2D). Upon stimulation with IFNg MHC-I expressionincreased in all HCC lines and was comparable for the WTand Erap1�/� TAgþ HCC of each HCC pair. Those two sets ofWT and Erap1�/� TAgþ HCC lines with IFNg-inducible APMwere used for further analysis.

ERAP1 regulates presentation of both IFNg-independent andIFNg-dependent tumor epitopes

Next, we examined the net effect of ERAP1on the recognition ofthe two MHC-I–restricted epitopes TAg-I and TAg-IV in depen-dence of IFNg . To do so, TCR gene-modified T cells recognizingeither TAg-I (TCR-I T cells) or TAg-IV (TCR-IV T cells) weregenerated by retroviral transduction of splenocytes from P14 xRag�/� donor mice (transduction rates were between 70% and90%). When TCR-I T cells were cocultured with unstimulatedWT and Erap1�/� TAgþHCC lines, WT TAgþHCCwas recognizedsignificantly better as compared with Erap1�/� TAgþ HCC(Fig. 3A). In contrast, upon stimulation with rmIFNg beforeTCR-I T cell coculture, the effect of ERAP1 was negligible asshown by comparably good recognition of WT and Erap1�/�

TAgþ HCC (Fig. 3B). These data reveal that TAg-I is IFNg-independent in the case of ERAP1-expression, but is IFNg-dependent in the absence of ERAP1.

C

B

F

A D

C4 E5NST

TAg-I

11mer

12mer

0

20

40

60

80

100

120

***

*

***

Pept

ide

trans

loca

tion

(%)

C4 E5NST

TAg-IV

9mer

10mer

0

20

40

60

80

100

120

******

***

***

Pep

tide

trans

loca

tion

(%)ADP

ATPADPATP

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

RVSAINNYAQKL(12mer)

SAINNYAQKL(TAg-I)

AINNYAQKL

VSAINNYAQKL(11mer) 0

3060

240

120

360

min

Retention time (min)Retention time (min)12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

DSVVYDFLKC(10mer)

SVVYDFLKC(9mer) 0

3060

240

120

360

Emin

Retention time (min)12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

VSAINNYAQKL(11mer)

SAINNYAQKL(TAg-I)

AINNYAQKL

0

30

60

240

120

360

min

Retention time (min)12 13 14 15 16 17 18 19 20 21 22 23 24

SVVYDFLKC(9mer)

VVYDFLKC(TAg-IV)

0

30

60

240

120

360

min

Figure 1.

Two MHC-I epitopes derived from thesame tumor antigen show varyinglystrong dependence on twoconsecutive steps of antigenprocessing. A, Transport assay forTAg-I and related N-terminallyextendedprecursor peptides. C4, high-affinity peptide devoid of an N-coreglycosylation site and labeled withfluorescein; E5, peptide not binding toTAP, including an N-core glycosylationsite and labeled with fluorescein; NST,reporter peptide, including an N-coreglycosylation site and labeled withfluorescein. The experimentalthreshold (red dotted line) was set forE5þATP (no TAP-binding). B and C,HPLC analysis of TAg-I-containingN-terminally extended precursorpeptides trimmed by recombinantmouse ERAP1 in vitro. 3–6 ng rmERAP1were incubated with 50 mmol/Lpeptide at 37�C and samples wereanalyzed by HPLC after 0 to 360minutes (label on the right). D,Transport assay for TAg-IV and relatedN-terminally extended precursorpeptides was performed as describedin A. E and F, HPLC analysis of TAg-IV-containing N-terminally extendedprecursor peptides trimmed byrecombinantmouse ERAP1 in vitro. Theexperiment was performed asdescribed for B and C. A and D, Dataare represented as mean � SD, two-way ANOVA with Bonferroni posttests(�� , P < 0.01; and ��� , P < 0.001).

ERAP1 Is Crucial for Peripheral T-cell Expansion

www.aacrjournals.org Cancer Res; 78(12) June 15, 2018 3245

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

In contrast with TAg-I, TAg-IV was recognized by TCR-IV T cellson unstimulated WT TAgþ HCC, but not on unstimulatedErap1�/� TAgþ HCC (Fig. 3C). IFNg stimulation before theTCR-IV T-cell coculture increased the recognition of TAg-IV inthe absence of ERAP1, but a significant difference wasmaintainedbetween WT and Erap1�/� TAgþ HCC (Fig. 3D). Hence, optimalpresentation and CTL recognition of TAg-IV critically requiredjoint action of IFNg and ERAP1.

Absence of ERAP1 accelerates tumor growth in immune-deficient recipients

In LoxP-Tag � Alb-Cre mice, WT and Erap1�/� TAgþ HCCprimarily developed in a host that was tolerant for their trans-

plantation rejection antigen (16, 17). Therefore, the immunoge-nicity of WT and Erap1�/� TAgþ HCC was tested in immune-competent mice. After subcutaneous transplantation into WT(Erap1þ/þ, C57BL/6) and Erap1�/� mice (18), the recipients wereobserved for a period of 100 days. Of 3 to 4 mice injected pergroup, none developed a tumor, because those recipients arecapable of priming a functional T-cell response toward TAg-I andTAg-IV (Supplementary Fig. S2A–S2C). To establish full-growntumors, WT and Erap1�/� TAgþ HCC were grown in immune-deficient Rag�/� mice (Fig. 4A; Supplementary S2A). Research byothers proposes a role of natural killer (NK) cells in recognizingERAP-deficient tumor cells (21, 22). Therefore, we comparedprogression of WT versus Erap1�/� TAgþ HCC in NK-cell–

A

Triple-transgenic mice developed WT or Erap1-/- TAg+ HCC

X

Erap1+/- x LoxP-Tag Erap1+/- x Alb-Cre

X

Erap1-/- LoxP-Tag

X

Alb-Cre Erap1-/-

B

Era

p1-/-

W

T

TAg Ki-67 H&E

D HCC pair 1

171 10

781 43

103

104

102

101

100 103 104 102 101 100

137 12

698 31

103

104

102

101

100 103 104 102 101 100

WT Erap1-/-

HCC pair 2

259 90

1653 342

103

104

102

101

100 103 104 102 101 100

WT

H2-Kb

H2-

Db

IFNγ + +

100 kDa ERAP1

β-Actin

TAP2

40 kDa

100 kDa

130 kDa

130 kDa

70 kDa

70 kDa

TAg

STAT1

pSTAT1

JAK1

JAK2

TAP1

LMP2 (ß1i)

LMP7 (ß5i)

MECL1 (ß2i)

- + - WT Erap1-/-

- + - WT Erap1-/-

25 kDa

25 kDa

25 kDa

100 kDa

100 kDa

HCC pair 1 HCC pair 2 C

200 8

1510 19

103

104

102

101

100 103 104 102 101 100

Erap1-/-

- IFNγ + isotype + IFNγ + isotype - IFNγ + antibody + IFNγ + antibody

MW

Figure 2.

Primary WT and Erap1�/� TAgþ HCC with IFNg-inducible antigen-processing machinery was generated. A, Breeding strategy to obtain triple-transgenicErap1þ/þ (WT)� LoxP-Tag� Alb-Cre and Erap1�/� � LoxP-Tag� Alb-Cre mice. B, Histologic analysis of paraffin-embedded primary WT and Erap1�/� TAgþ HCCof 3 to 4 months old LoxP-Tag � Alb-Cre mice. One representative example is shown. C, Western blot analysis of IFNg-dependent expression of antigenprocessing machinery components in WT and Erap1�/� TAgþ HCC. D, FACS analysis of MHC-I expression in WT and Erap1�/� TAgþ HCC.

Schmidt et al.

Cancer Res; 78(12) June 15, 2018 Cancer Research3246

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

competent (Rag�/�) recipients, progression of WT TAgþ HCC inNK-cell–competent versus NK-cell–deficient (Rag�/� x gc�/�)recipients, and progression of Erap1�/� TAgþ HCC in NK-cell–competent versus NK-cell–deficient recipients. No impact of NKcells on tumor growth of TAgþHCC could be seen in themajorityof mice (Supplementary Fig. S3A–S3C). One mouse showedexceptional tumor outgrowth of Erap1�/� TAgþ HCC starting50 days after tumor cell inoculation. But NK-cell contribution atthis late stage is unlikely.We concluded thatNK-cell cytotoxicity isnot relevant in the herein usedmodel of transplanted TAgþHCC.

Interestingly, when the tumor size was compared in depen-dence of recipient's ERAP1 at the day of ATT,WT TAgþHCC had amean volume of 92 mm3 (WT!WT SD � 63.5) in Rag�/�

recipients while having a mean volume of 118 mm3

(WT!Erap1�/� SD � 57.9) in Erap1�/� x Rag�/� recipients.Similarly, Erap1�/� TAgþ HCC were on average 143 mm3 inRag�/� recipients (Erap1�/�!WT SD � 62.3) as compared withErap1�/� TAgþ HCC having a mean volume of 233 mm3

(Erap1�/�!Erap1�/� SD � 123.3; Supplementary Fig. S3D).In conclusion, lack of ERAP1 the noncancerous cells ofimmune-deficient recipients accelerated tumor growth aftertransplantation.

ERAP1 facilitates efficient rejection of TAgþHCC through TCR-IT cells

The use of TCR gene-modified T cells is a particularly effectiveapproach to target malignancies (23). To compare the impact ofERAP1-dependent direct presentation, MHC-I–dependent cross-presentation, and ERAP1-dependent cross-presentation on ATT

efficiency, WT or Erap1�/� TAgþ HCCwere established in ERAP1-competent H2b recipients (Rag�/�, WT/H2b), ERAP1-competentMHC-I–mismatched recipients (SCID, WT/H2d), and ERAP1-deficient H2b recipients (Rag�/�, Erap1�/�/H2b; Fig. 4A). At first,we investigated whether ERAP1 specifically affected rejection ofTAgþ HCC through TCR-I T cells targeting TAg-I. The highestrejection rate was observed, when WT TAgþ HCC were treated inWT/H2b recipientswith 100%and88%ofWTTAgþofHCCpair 1and 2 being rejected, respectively (Fig. 4B; Supplementary TableS1A). Notably, TCR-I gene-modified T cells were not capable ofrejecting WT TAgþ HCC in WT/H2d recipients, where the targetantigen cannot be cross-presented via MHC-I. Reduced rejectionrates of 67% and 57% were also observed, when WT TAgþ HCCwas treated with TCR-I T cells in Erap1�/�/H2b recipients. Incontrast with WT TAgþ HCC, a reduced number of 70% and80% Erap1�/� TAgþ HCC was rejected in WT/H2b recipients,indicating some contribution of ERAP1 on the direct presentationof TAg-I in vivo. Although Erap1�/� TAgþHCC was not rejected inWT/H2d recipients, HCC pair 1 and 2 showed a huge differencewith 33% and 100%being rejected, respectively, in the absence ofhost ERAP1 (Fig. 4B; Supplementary Table S1A). Hence, althoughexpression ofMHC-I and ERAP1 in ATT recipient's cells seemed tobe required for rejection through TCR-I T cells, ERAP1-dependentdirect presentation of TAg-I exerted a moderate effect.

Rejection of TAgþ HCC through TCR-IV T cells criticallydepends on ERAP1 expression in noncancerous cells

Adoptive transfers targeting TAg-IV through TCR-IV T cells wereconducted in parallel. Here, in the group of WT/H2b recipients,

A

B D

C

1 10 100 1,000 0

5,000

10,000

15,000

20,000

25,000 WT Erap1 -/-

HCC pair 1

E:T Ratio 1 10 100 1,000

0

5,000

10,000

15,000

20,000

25,000 WT Erap1 -/-

HCC pair 2

E:T Ratio 1 10 100 1,000

0

5,000

10,000

15,000

20,000 WT Erap1 -/-

HCC pair 2

E:T Ratio

***

**

1 10 100 1,000 0

5,000

10,000

15,000

20,000 WT Erap1 -/-

HCC pair 2

E:T Ratio

***

1 10 100 1,000 0

5,000

10,000

15,000

20,000 WT Erap1 -/-

HCC pair 1

E:T Ratio

**

**

1 10 100 1,000 0

5,000

10,000

15,000

20,000 WT Erap1 -/-

HCC pair 1

E:T Ratio

**

-IFNγ/TCR-IV -IFNγ/TCR-I

+IFNγ/TCR-IV +IFNγ/TCR-I

1 10 100 1,000 0

5,000

10,000

15,000

20,000

25,000 WT Erap1 -/-

HCC pair 2

***

**

E:T Ratio 1 10 100 1,000

0

5,000

10,000

15,000

20,000

25,000 WT Erap1 -/-

HCC pair 1

**

**

E:T Ratio

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)IF

Nγ

(pg/

mL)

IFN

γ (p

g/m

L)IF

Nγ

(pg/

mL)

IFN

γ (p

g/m

L)IF

Nγ

(pg/

mL)

IFN

γ (p

g/m

L)

Figure 3.

ERAP1 regulates presentation of the subdominant epitope TAg-I and dominant epitope TAg-IV on TAg-driven primary HCC. A and B, Coculture ofTAg-I–specific TCR-I T cells with unstimulated (A) and IFNg-stimulated (B) WT and Erap1�/� TAgþ HCC. C and D, Coculture of TAg-IV-specific TCR-IVT cells with unstimulated (C) and IFNg-stimulated (D) WT and Erap1�/� TAgþ HCC. A–D, Data are represented as mean � SD, two-way ANOVAwith Bonferroni posttests (�� , P < 0.01; ��� , P < 0.001).

ERAP1 Is Crucial for Peripheral T-cell Expansion

www.aacrjournals.org Cancer Res; 78(12) June 15, 2018 3247

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

complete rejection was observed for WT TAgþ HCC (Fig. 4C;Supplementary Table S1B). WT TAgþ HCC was not rejected ifantigen cross-presentation was lacking in WT/H2d mice andstrikingly, WT TAgþ HCC rejection rates were also stronglyreduced (33% and 14%) if ERAP1 was not expressed in therecipient's cells. In addition, ERAP1 affected direct presentationof TAg-IV, as indicated by inferior rejection of 70% and 83%Erap1�/� TAgþ HCC in WT/H2b recipients. The requirement ofantigen cross-presentation of TAg-IV was confirmed by lack ofrejection of Erap1�/� TAgþHCC inWT/H2dmice, and by less thanhalf (45%) of the Erap1�/� TAgþHCC being rejected in Erap1�/�/H2b recipients (Fig. 4C; Supplementary Table S1B). In summary,ERAP1-depedent direct presentation moderately affected tumorrejection, whereas both, MHC-I–dependent antigen cross-presen-tation and ERAP1 expression in noncancerous cells were criticallyrequired for rejection of TAgþ HCC through TCR-IV gene-mod-ified cells (Fig. 4D).

Absence of ERAP1 enables escape of small TAgþ HCC despitethe presence of functional CTL

Next, tumor volumes at the day of ATT were correlated withsubsequent tumor rejection to rule out that the above described

variations between the groups affected the efficiency of TCR-Ior TCR-IV T-cell therapy. At first, 57 mice treated with TCR-IT cells were analyzed (Supplementary Fig. S3E). Mice weregrouped according to whether tumors were rejected or notand the tumor size at the day of treatment was plotted forboth groups. In the case of TCR-I, both rejected and nonrejectedtumors had an average volume of 132 mm3 and 135 mm3,respectively (rejected SD � 92.2; not rejected SD � 95.4;Fig. 5A). The same analysis was performed for 57 mice treatedwith TCR-IV T cells (Supplementary Fig. S3F). In this case,rejected tumors had a mean volume of 135 mm3 on the day ofATT, whereas nonrejected tumors were on average 171 mm3

(rejected SD � 83.9; not rejected SD � 104.2; Fig. 5B). Insummary, the tumor size at the day of ATT did not affectrejection through TCR-I T cells or TCR-IV T cells.

In addition, T lymphocyte function was analyzed by in vivocytotoxicity analysis at the end of the experiments. Naive controlmice did not respond to TAg-I, that is, they did not kill TAg-I(SAINNYAQKL)-loaded target cells, whereas the same peptide-loaded target cells were efficiently killed by TAg-immunized miceor TCR-I T-cell–treated mice (Fig. 5C). TAg (16.113)-immunizedcontrols showed amean of 75% specific cytotoxicity toward TAg-I

A

B C

TCR-I

0% 100% Rejection

Tumor Erap1

WT

WT

-/-

WT

-/-

-/-

Recipient‘s Erap1

WT

WT

WT

-/-

WT

-/-

Recipient‘s haplo- type

H2b

H2d

H2b

H2b

H2d

H2b

HCC pair 1

100

0.0

70.0

66.7

0.0

33.3

HCC pair 2

87.5

n.a.

80.0

57.1

n.a.

100

TCR-IV

0% 100% Rejection

Tumor Erap1

WT

WT

-/-

WT

-/-

-/-

Recipient‘s Erap1

WT

WT

WT

-/-

WT

-/-

Recipient‘s haplo- type

H2b

H2d

H2b

H2b

H2d

H2b

HCC pair 1

100

0.0

70.0

33.3

0.0

50.0

HCC pair 2

100

n.a.

83.3

14.3

n.a.

40.0

D

WT or Erap1-/- TAg+ HCC

Tumor transplantation

Monitoring of tumor rejection

~30–100 days ~50–100 days

Tumor growth

T-cell transfer

Immune-deficient recipients

0 20 40 60 80 100

TumorErap1

Recipient'sErap1

WT

WT

WT

WT-/-

-/-

-/-

-/-

*

WT

WT

WT

WT-/-

-/-

-/-

-/-

93.8

61.9

75.0

66.7

100

23.8

76.7

45.0

Rejection (%)

Figure 4.

T-cell–mediated rejection of TAgþ HCC requires antigen cross-presentation and ERAP1. A, Schematic for tumor transplantation and rejection experiments. B,Graphical representation of rejection of WT and Erap1�/� TAgþ HCC by TCR-I T cells in immune-deficient recipients. For numerical summary, see SupplementaryTable S1A. C, Graphical representation of rejection of WT and Erap1�/� TAgþ HCC by TCR-IV T cells in immune-deficient recipients. For numerical summary, seeSupplementary Table S1B. B and C, Numbers represent the percentage of rejection. D, Summary of the percentage of rejection of HCC pair 1 and 2 in the H2b

recipient groups is depicted in B and C. Numbers are the percentage of rejection. Data are represented as mean of HCC pair 1 and 2 � SD, two-wayANOVA with Bonferroni posttests (� , P < 0.05).

Schmidt et al.

Cancer Res; 78(12) June 15, 2018 Cancer Research3248

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

(SD � 18.0). For a determination of possible differences in thefunctionality of persistent CTLs after tumor rejection or recur-rence,mice treatedwith TCR-I T cellswere groupedonwhether theTAgþ HCC were rejected or not. But while mice rejecting throughTCR-I showed 87% cytotoxicity in vivo (SD � 21.3), a mean of81% TAg-I–specific cytotoxicity was detectable in mice withnonrejected tumors as well (SD � 30.2). These data confirmedthat the functionality of TCR-I T cells was maintained throughoutthe experiment, and failure of rejectionwas not caused by a loss offunction of the transferred TCR-I T cells (Fig. 5D). Similarly, uponanalysis of adoptively transferred TCR-IV T cells, naive controlmice did not kill TAg-IV (VVYDFLKL)–loaded target cells, whereasTAg (16.113)-immunized control mice presented on average94% (SD � 5.8) TAg-IV–specific cytotoxicity. In the samplegroups, on average 89% (SD� 20.7) TAg-IV–specific cytotoxicitywas detectable in those mice in which TAgþ HCC were rejected,whereas mice with nonrejected TAgþ HCC showed a mean of88% (SD � 21.6) TAg-IV–specific cytotoxicity (Fig. 5E). In sum-mary, rejection of TAgþ HCC failed despite the presence of in vivocytotoxic TCR-IV T cells.

Expansion of TCR gene-modified T cells requires ERAP1expression in ATT recipients

For ATT, therapeutic T cells are transferred into lympho-deplet-ed individuals, because enhancedhomeostatic T-cell proliferation

is required for efficient antitumor immunity (24, 25). Hence, theexpansion of CD8þ T cells (within white blood cells) was ana-lyzed and compared between the different recipient groups (Sup-plementary Fig. S4A–S4C). In the case of TCR-I T cells, CD8þ cellsconstituted on average 0.5% (SD � 0.75) in WT/H2b recipientsbearing WT TAgþ HCC one week after ATT. Similarly, in WT/H2b

recipients bearingErap1�/�TAgþHCCapproximately 0.6%(SD�0.89)were CD8þ T cells (Fig. 6A). In contrast, TCR-I T cells did notexpand in WT/H2d recipients and were barely detectable inErap1�/�/H2b recipients regardless of whether they were bearingWT TAgþHCC or Erap1�/� TAgþHCC (Fig. 6A). At this early timepoint a considerable variation was observed between mice, andMHC-I–independent proliferation of TCR-I T cells was noticed inthe WT/H2d group bearing Erap1�/� TAgþ HCC. Control experi-ments revealed that in vitro primed TCR-I gene–modified T cellsdid not expand in tumor-free recipients in the absence of TAg,whereas in vivo primed TAg-I–specific TE cells were capable ofexpanding antigen-independently (Supplementary Fig. S5A andS5B). Fourweeks after ATT, a sustained expansionwas observed inall WT/H2b recipients, where CD8þ T cells now constituted amean of 9% (SD� 6.57) and 7% (SD� 3.90) inmice bearingWTTAgþ HCC or Erap1�/� TAgþ HCC, respectively (Fig. 6B). Still,CD8þ T cells were not detectable in WT/H2d mice and were verylow in Erap1�/�/H2b recipients reaching a mean of 1% (SD �0.83) and 3% (SD � 3.51; Fig. 6B). In summary, expansion of

ED

B CANaïve control Immunized control

Rejected Not rejected

1.3

51.648.4

68.731.3

97.82.2

90.19.9

UnloadedTAg-I

UnloadedTAg-I

UnloadedTAg-I

UnloadedTAg-I

CFSE103 104102101100

1K

800

600

400

200

0

SS

C-H

Cou

nts

CFSE103 104102101100

CFSE103 104102101100

CFSE103 104102101100

CFSE103 104102101100

Cou

nts

Cou

nts

Cou

nts

0

200

400

600

ns

RejectedNot

rejected

Tum

or v

olum

e (m

m3 )

Tum

or v

olum

e (m

m3 )

0

200

400

600

ns

RejectedNot

rejected

0

20

40

60

80

100

TAg-I

Spe

cific

kill

in v

ivo

(%)

Naïvecontrol Rejected

Notrejected

Immunizedcontrol

P = 0.0004

0

20

40

60

80

100

TAg-IV

Naïvecontrol Rejected

Notrejected

Immunizedcontrol

Spe

cific

kill

in v

ivo

(%)

P = 0.0002

Figure 5.

Small TAgþ HCC recur in the presence of epitope-specific functional cytotoxic T lymphocytes. A, All mice depicted in Supplementary Fig. S3E weregrouped according to whether TAgþ HCC was rejected or not and the tumor volume at the day of ATT was plotted, rejected (n ¼ 43), not rejected (n ¼ 14).B, All mice depicted in Supplementary Fig. S3F were grouped according to whether TAgþ HCC was rejected or not and the tumor volume at the dayof ATT was plotted, rejected (n¼ 37), not rejected (n¼ 20). A and B, All data are represented with mean� SD, Mann–Whitney test; � , P < 0.05. C and D, In vivocytotoxicity analysis for TAg-I. To detect CTL activity in vivo, TAg-I and/or TAg-IV–loaded spleen cells were labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE) and were injected into the indicated mice. The ratio between different CFSE-labeled populations was determined and18 hours later by flow cytometry. One representative example for TAg-I is shown in C and data of three experiments are shown in D. Na€�ve control,n ¼ 6; immunized control, n ¼ 6; rejected, n ¼ 24; not rejected (n ¼ 6). E, In vivo cytotoxicity analysis for TAg-IV was performed as described in C. Data ofthree experiments are shown. Na€�ve control, n ¼ 8; immunized control, n ¼ 8; rejected, n ¼ 22; not rejected, n ¼ 8. D and E, All data are represented withmean � SD, Kruskal–Wallis test.

ERAP1 Is Crucial for Peripheral T-cell Expansion

www.aacrjournals.org Cancer Res; 78(12) June 15, 2018 3249

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

adoptively transferred gene-modified TCR-I T cells required com-patible MHC-I, and expression of ERAP1 in ATT recipients,whereas lack of ERAP1 in cancerous cells had not impact hereon.

Expansion of TCR-IV T cells was analyzed at the same time.Here, too, a strong variation was observed between individualmice one week after ATT. On average, 0.8% (SD � 1.64) to 0.9%

(SD � 1.20) CD8þ T cells were detectable in WT/H2b recipientsbearing WT or Erap1�/� TAgþ HCC (Fig. 6C). TCR-IV T cellsdid not expand in WT/H2d mice and represented less than0.3% in Erap1�/�/H2b recipients (Fig. 6C). Similar to TCR-I Tcells, TCR-IV T cells expanded within the following 3 weeks.Accordingly, 4 weeks after ATT a mean of 5% (SD � 1.57) and

A B

DC

E

0

1

2

3

4 *

TCR-I/1 week

Rejected Not

rejected

CD

8+ T c

ells

(%)

CD

8+ T c

ells

(%)

CD

8+ T c

ells

(%)

CD

8+ T c

ells

(%)

F

0

5

10

15

20

25ns

TCR-I/4 weeks

RejectedNot

rejected

G

0

2

4

6

8*

TCR-IV/1 week

RejectedNot

rejected

H

0

5

10

15

20

25*

TCR-IV/4 weeks

RejectedNot

rejected

0

1

2

3

4P < 0.01

TCR-I/1 week

WT WT -/--/- WT-/-

-/-WT

Tumor Erap1Recipient's Erap1

Recipient's haplotype H2b H2dH2b H2bH2d H2bWTWT -/-WT

CD

8+ T c

ells

(%)

0

5

10

15

20

25P < 0.01

TCR-I/4 weeks

WT WT -/--/- WT-/-

-/-WTH2b H2dH2b H2bH2d H2b

WTWT -/-WTTumor Erap1

Recipient's Erap1Recipient's haplotype

CD

8+ T c

ells

(%)

0

2

4

6

8P < 0.001

TCR-IV/1 week

WT WT -/--/- WT-/-

-/-WTH2b H2dH2b H2bH2d H2b

WTWT -/-WTTumor Erap1

Recipient's Erap1Recipient's haplotype

CD

8+ T c

ells

(%)

0

5

10

15

20

25P < 0.001

TCR-IV/4 weeks

WT WT -/--/- WT-/-

-/-WTH2b H2dH2b H2bH2d H2b

WTWT -/-WTTumor Erap1

Recipient's Erap1Recipient's haplotype

CD

8+ T c

ells

(%)

Figure 6.

ERAP1 expression in noncancerous cells supports immediate expansion of epitope-specific TCR-I and TCR-IV T cells and decides on tumor rejection.A, FACS analysisof TCR-I T-cell expansion oneweek after ATT.WT!WT/H2b, n¼ 16;WT!WT/H2d, n¼ 3;WT!Erap1�/�/H2b, n¼ 11; Erap1�/�!WT/H2b, n¼ 7; Erap1�/�!WT/H2d,n¼4;Erap1�/�!Erap1�/�/H2b,n¼5.B,FACSanalysis of TCR-I T cell expansion fourweeks afterATT.WT!WT/H2b,n¼6;WT!WT/H2d,n¼ 2;WT!Erap1�/�/H2b,n ¼ 3; Erap1�/�!WT/H2b, n ¼ 9; Erap1�/�!WT/H2d, n ¼ 4; Erap1�/�!Erap1�/�/H2b, n ¼ 7. C, FACS analysis of TCR-IV T cells performed one week after ATT.WT!WT/H2b, n ¼ 16; WT!WT/H2d, n ¼ 4; WT!Erap1�/�/H2b, n ¼ 11; Erap1�/�!WT/H2b, n ¼ 12; Erap1�/�!WT/H2d, n ¼ 3; Erap1�/�!Erap1�/�/H2b, n ¼ 6. D,FACS analysis of TCR-IV T cells four weeks after ATT. WT!WT/H2b, n ¼ 6; WT!WT/H2d, n ¼ 3; WT!Erap1�/�/H2b, n ¼ 4; Erap1�/�!WT/H2b, n ¼ 10;Erap1�/�!WT/H2d, n¼ 3;Erap1�/�!Erap1�/�/H2b, n¼6.A–D,Data of n¼ 3 experiments are shownwithmean� SD, Kruskal–Wallis test.E,Allmicewith haplotypeH2b depicted inAwere grouped according to whether TAgþ HCCwas rejected or not and the percentage of CD8þ T cells was plotted, rejected (n¼ 28), not rejected(n ¼ 11). F, All mice with haplotype H2b depicted in B were grouped according to whether TAgþ HCC was rejected or not and the percentage of CD8þ T cellswas plotted, rejected (n ¼ 21), not rejected (n ¼ 4). G, All mice with haplotype H2b depicted in C were grouped according to whether TAgþ HCC was rejected ornot and the percentage of CD8þ T cells was plotted, rejected (n ¼ 30), not rejected (n ¼ 15). H, All mice with haplotype H2b depicted in D were groupedaccording to whether TAgþ HCC was rejected or not and the percentage of CD8þ T cells was plotted, rejected (n ¼ 19), not rejected (n ¼ 7). E–H, Data arerepresented with mean � SD, Mann–Whitney test (� , P < 0.05).

Schmidt et al.

Cancer Res; 78(12) June 15, 2018 Cancer Research3250

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

8% (SD� 8.11) CD8þ T cells were detected inWT/H2b recipientsbearing WT or Erap1�/� TAgþ HCC, respectively (Fig. 6D). InWT/H2d recipients or Erap1�/�/H2b recipients, CD8þ T cells stillconstituted no more than 0.01% to 0.8% (Fig. 6D). AlthoughTAg-I–specific T cells may also expand antigen independently,TAg-IV–specific T cells expanded exclusively in the presenceof TAg (Supplementary Fig. S5C). Interestingly, if polyclonalTAg-specific CD8þ effector T (TE) cells [containing a mean of1.6% (SD � 0.93, n ¼ 3) TAg-I–specific TE cells and 8.4% (SD �7.89, n ¼ 3) TAg-IV–specific TE cells] were isolated fromTAg-immunized C57BL/6 mice, and were transferred into TAgþ

HCC-bearing recipients, ERAP1 assumed a more prominentrole on tumor rejection as related to direct epitope presentation.However, overall expansion of the adoptively transferredpolyclonal CD8þ TE cells was not affected in ERAP1-deficientrecipients, indicating that only antigen-specific T cells requiredrecipient's ERAP1 to proliferate (Supplementary Fig. S6A–S6D).In conclusion, the recipients' ERAP1 status, but not the ERAP1status of the targeted TAgþ HCC was crucial for expansion ofepitope-specific TCR gene-modified T cells.

Immediate expansion of antigen-specific T cells decides ontumor rejection

Impaired expansion of antigen-specific T cells after ATT was aremarkable symptom of ERAP1-deficient ATT recipients, inwhich TAgþ HCC was poorly rejected. To examine whetherexpansion of TCR-I T cells correlated with rejection of TAgþ

HCC, all H2b recipient mice that were analyzed according totheir percentage of TCR-I CD8þ T cells in the blood one weekafter ATT were grouped on whether mice had rejected the TAgþ

HCC or not. At this time point mice with later on rejectedtumors presented a mean of 0.5% (SD � 0.73) CD8þ T cells ascompared with a significantly lower mean of 0.1% (SD � 0.19)CD8þ T lymphocytes in mice that subsequently did not rejecttumors (Fig. 6E). When the H2b TCR-I T-cell–treated mice weregrouped according to the same parameters, CD8þ T cellsconstituted on average 6% (SD � 4.92) in mice with later onrejected tumors versus a mean of 4% (SD � 6.60) CD8þ T cellsin those mice where tumors were not rejected (Fig. 6F). Hence,immediate expansion of TCR-I T cells one week after ATTcorrelated with subsequent tumor rejection.

In those mice adoptively transferred with TCR-IV T cells,approximately 0.8% (SD � 1.47) CD8þ T lymphocytes weredetectable one week after ATT in H2b mice that rejected TAgþ

HCC later on.Opposite to that, a significantly lower percentage ofapproximately 0.1% (SD � 0.14) CD8þ T cells was detected inmice with subsequently nonrejected TAgþ HCC (Fig. 6G). Thatstatistical connection was still observable 4 weeks after ATT, andon average 6%(SD�6.56)CD8þT cells were present in the bloodof the mice with ultimately rejected tumors. In contrast, thosemice that did not reject tumors had a mean of 0.8% (SD � 0.79)of CD8þ T cells (Fig. 6H). These data confirm that both, ERAP1function in ATT recipients and T-cell expansion and persistence,are required for successful tumor rejection after ATT.

Lack of ERAP1 enables escape of T-cell–recognizable IFNg-responsive TAgþ HCC

Relating to earlier observations in Textor and colleagues (12) ofIFNg–unresponsive cancer variants to evade T-cell recognition,we wanted to rule out that an acquired deficiency in IFNg-signaling contributed to TAgþ HCC recurrence within the context

of ERAP1-regulated epitope presentation. Six nonrejected TAgþ

HCCwere grown in vitro for further analysis. Among these, twoWTTAgþ HCC were reisolated after TCR-I T-cell therapy in Erap1�/�

recipients (#377, #378), one WT TAgþ HCC was reisolated afterTCR-IV T-cell therapy in an Erap1�/� recipient (#400), oneErap1�/� TAgþ HCC was reisolated after TCR-IV T-cell therapyin an Erap1�/� recipient (#556), and two Erap1�/� TAgþ HCCwere reisolated after TCR-IV T cell therapy in ERAP1-competentrecipients (#306, #282, Fig. 7A). First, we confirmed expression ofthe transplantation rejection antigen TAg by the nonrejectedHCC(Fig. 7B). After stimulation with rmIFNg , JAK1 was constitutivelyexpressed in all reisolated TAgþ HCC lines in different amounts,but upregulation of STAT1, as well as phosphorylation thereof,was always detectable (Fig. 7B). In addition, TAP1 and TAP2 werehighly expressed in all six TAgþHCCafter IFNg stimulation, just asthe immunoproteasome subunits b1i, b5i, and b2i (Fig. 7B).When MHC-I expression was analyzed, all reisolated tumorsshowed expression of both alleles, H2-Db and H2-Kb, albeit withvarying degree (Fig. 7C).

Finally, in vitro recognition of the reisolated tumor cells byTCR-I and TCR-IV T cells was analyzed. All six TAgþ HCCs wererecognized by TCR-I T cells (Fig. 7D). Likewise, all six TAgþ HCCwere well recognized by TCR-IV T cells, with the exceptionof #556, the Erap1�/� TAgþ HCC that has progressed afterTCR-IV T-cell therapy in an Erap1�/� recipient. This HCC lineshowed an IFNg-inducible, albeit very low, CTL response that wasin accordance with the rather low expression of MHC-I by thisHCC line in comparison with the other nonrejected TAgþ HCC(Fig. 7E). If antigen cross-presentation was assessed in bonemarrow–derived dendritic cells (BMDC), a slightly impairedcross-presentation of TAg-I and TAg-IV as processed from full-lengthpurifiedTAgproteinwasobservedwithErap1�/�BMDCs incomparison with WT BMDCs (Supplementary Fig. S7A and S7B).We concluded that the sole lack of ERAP1 in tumor cells or in therecipient's non-tumor cells determined the failure of ATT, eventhough further experimentation is required to elucidate the actualantigen cross-presentation pathway involving ERAP1.

DiscussionWe showed that ERAP1 is critically required for the processing

of MHC-I epitopes as targeted by ATT and critically affects suc-cessful eradication of cancer. In this study, rejection of transplant-able tumors was decreased by 25% and 23% for TCR-I andTCR-IV, respectively, if cancer cells were deficient for ERAP1 butexpressed other IFNg-inducible APM components such as immu-noproteasomes and TAP. The previously reported inference ofTAg-I being ERAP1-independently processed was limited to directantigen presentation in the presence of IFNg and with cross-presented TAg (12). The herein presented data imply that noother APM components are capable of completely substitutingthe specialized function of ERAP1.

Previously, it was shown by others that NK cells can rejectsuspensions of ERAP1-silenced RMA lymphoma cells due to poorengagement of the Ly49C/INK-cell–inhibitory receptor by alteredpMHC-I in the absence of N-terminal trimming of peptides (21).And pharmacological inhibition of ERAP1 in human tumor cellsinduced anNK-cell response causedby the tumor cell's inability toengage inhibitory NK-cell receptors (22). Adoptive T-cell therapythrough transfer of TCR gene-modified T cells, however, is anapproach to target solid tumors, that escaped the patient's

ERAP1 Is Crucial for Peripheral T-cell Expansion

www.aacrjournals.org Cancer Res; 78(12) June 15, 2018 3251

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

spontaneous immune recognition and control experiments in thisstudy did not confirm an early role of NK cells, if Erap1 wasgenetically depleted and surface expression of MHC-I was adjust-able by IFNg . Here, the therapeutic avenue of pharmacologicallyinhibiting ERAP1 is certainly a strategy that contrasts adoptive T-cell transfer, if the latter one targets ERAP1-dependent MHC-Iepitopes. Another study showed that attenuation of ERAP1 func-

tion may induce cell surface presentation of protective tumorantigens inducing functional T-cell responses (11). Thosefindingsshow that enhancing ERAP1 function is probably not an alterna-tive therapeutic avenue either, because ERAP1 has the potential toover-trimpeptides and to destroyMHC-I epitopes. Our data are ofparticular importancewith regard to the various common SNPs inthe human Erap1 homolog and the resulting naturally existing

D

A

Stat1

pStat1 Long exposure

pStat1 Short exposure

β-Actin

IFNγ - +

JAK1

TAg

WT (HCC pair 2)

Recurred TAg+ HCC

- + - + - + - + - + - +

TAP1

TAP2

ERAP1

LMP2 (β1i)

LMP7 (β5i)

MECL1 (β2i)

Label #377 #378 #556 #306 #282 #400

100 kDa

40 kDa

100 kDa

130 kDa

70 kDa

70 kDa

25 kDa

25 kDa

25 kDa

100 kDa

100 kDa

100 kDa

CWT

162 34

215 40

103

104

102

101

100 103 104 102 101 100

Erap1-/-

210 18

415 22

103

104

102

101

100 103 104 102 101 100

#377 409 41

931 52

103

104

102

101

100 103 104 102 101 100

#556 68 11

95 11

103

104

102

101

100 103 104 102 101 100

#378 101 7

186 9

103

104

102

101

100 103 104 102 101 100

#306 157 47

189 37

103

104

102

101

100 103 104 102 101 100

#400 299 36

531 38

103

104

102

101

100 103 104 102 101 100

#282 506 590

552 559

103

104

102

101

100 103 104 102 101 100

H2-Kb

H2-

Db

- IFNγ + isotype + IFNγ + isotype

- IFNγ + antibody + IFNγ + antibody

B

Label #377 #378 #400 #556 #306 #282

Therapy TCR-I TCR-I TCR-IV TCR-IV TCR-IV TCR-IV

Tumor Erap1 WT WT WT -/- -/- -/-

Recipient’s Erap1 -/- -/- -/- -/- WT WT

E

0

25,000

50,000

75,000 TCR-I

-IFNγ +IFNγ -IFNγ +IFNγ

none WT #377 Erap1 -/- #378 #400 #556 #306 #282 Target cells

0

700

25,000

50,000

75,000 TCR-IV

none WT #377 Erap1 -/- #378 #400 #556 #306 #282 Target cells

IFN

γ (p

g/m

L)

IFN

γ (p

g/m

L)

MW

Figure 7.

Lack of ERAP1 enables recurrence of TAgþ, TCR-recognizable HCC with IFNg-inducible antigen-processing machinery. A, Overview of WT or Erap1�/� TAgþ HCCreisolated after TCR-I or TCR-IV therapy in WT/H2b or Erap1�/�/H2b recipients. B, Western blot analysis of IFNg-inducible APM components in reisolated TAgþ

HCC. C, FACS analysis of IFNg-inducible MHC-I expression for reisolated TAgþ HCC. D, TCR-I T-cell recognition of reisolated TAgþ HCC. E, TCR-IV T-cellrecognition of reisolated TAgþ HCC. D and E, Data are represented as mean � SD.

Schmidt et al.

Cancer Res; 78(12) June 15, 2018 Cancer Research3252

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

alleles of Erap1 encoding ERAP1 variants with different, some-times impaired, function (26–28). In this study, we used amousemodel of genetically depleted Erap1, causing complete loss offunction of the enzyme, and transferability of such findings tofunctionally impaired ERAP1 variants in humans remains to beseen. Nevertheless, personalized cancer immunotherapy of thefuture may consider processing of the targeted epitope as wellas patient's Erap1 haplotype as an additional marker. Furtherinvestigations, including MHC-I epitopes presented by clini-cally relevant tumor samples, would also show whether ourfindings, as obtained from a transplantable tumor model, canbe translated to improve adoptive T-cell therapy approaches inpatients.

For the first time, we show that ERAP1 is certainly playing a rolein antigen cross-presentation of tumor epitopes as targeted byATTand thereby supports rejection of established tumors. Previouswork by others reported that ERAP1 is required for cross-presen-tation of the model antigen OVA (13–15). But if tumor antigensare cross-presented in vivo, cell-associated antigens from dyingtumor cells may be the primary source of antigen. So far, only oneother study has examined ERAP1-dependent cross-presentationof cell-associated antigens. The cross-presentation of cell-associ-ated OVA and cell-associated male HY antigen was analyzed afterimmunizationof ERAP1-deficientmicewithMHC-I–mismatchedantigen-positive cells (15). Here, ERAP1-deficiency reduced pro-liferation of OVA-specific or HY antigen-specific CD8þ T cells,respectively. Our in vivo investigations with the tumor rejectionantigen TAg confirm these earlier observations, but analysis ofERAP1-dependent cross-presentation of TAg-I and TAg-IV inBMDCs in vitro requires improved experimental procedures.Importantly, our experiments showed that ERAP1 has a decisiveeffect on tumor rejection by being critically required for the

proliferation of CD8þ T cells after ATT, and that lack of ERAP1in ATT recipients consequently caused failure of adoptive T-celltherapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K. Schmidt, U. Seifert, T. Blankenstein, G. Willimsky,P.-M. KloetzelDevelopment of methodology: K. Schmidt, G. WillimskyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Schmidt, A. Textor, G. WillimskyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Schmidt, A. TextorWriting, review, and/or revision of the manuscript: K. Schmidt, A.A. K€uhl,A. Textor, T. Blankenstein, G. Willimsky, P.-M. KloetzelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Keller, A.A. K€uhlStudy supervision: K. Schmidt, G. Willimsky, P.-M. Kloetzel

AcknowledgmentsThe authors would like to thank F. Kirschner, A. Lehmann, and N. Albrecht-

K€opke from the Institute of Biochemistry, Charit�e—Universit€atsmedizin Berlinfor assistancewith data acquisition.U. Seifert, T. Blankenstein, andG.Willimskyreceived grants from the Deutsche Forschungsgemeinschaft (SFB-TR36).T. Blankenstein and P.-M. Kloetzel received grants from the Berlin Instituteof Health (CRG-1). P.-M. Kloetzel received funding from the Wilhelm Sander-Stiftung (2015.107.1).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 29, 2017; revised February 13, 2018; acceptedMarch 16, 2018;published first March 20, 2018.

References1. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ,

et al. Durable complete responses in heavily pretreated patients withmetastatic melanoma using T-cell transfer immunotherapy. Clin CancerRes 2011;17:4550–7.

2. Spiotto MT, Yu P, Rowley DA, Nishimura MI, Meredith SC, Gajewski TF,et al. Increasing tumor antigen expression overcomes "ignorance" to solidtumors via crosspresentation by bone marrow-derived stromal cells.Immunity 2002;17:737–47.

3. SpiottoMT, Rowley DA, SchreiberH. Bystander elimination of antigen lossvariants in established tumors. Nat Med 2004;10:294–8.

4. Leisegang M, Engels B, Schreiber K, Yew PY, Kiyotani K, Idel C, et al.Eradication of large solid tumors by gene therapy with a T-cell receptortargeting a single cancer-specific point mutation. Clin Cancer Res 2016;22:2734–43.

5. Engels B, Engelhard VH, Sidney J, Sette A, Binder DC, Liu RB, et al. Relapseor eradication of cancer is predicted by peptide-major histocompatibilitycomplex affinity. Cancer Cell 2013;23:516–26.

6. Robbins PF, Dudley ME, Wunderlich J, El-Gamil M, Li YF, Zhou J, et al.Cutting edge: persistence of transferred lymphocyte clonotypes correlateswith cancer regression in patients receiving cell transfer therapy. J Immunol2004;173:7125–30.

7. Kloetzel PM. Antigen processing by the proteasome. Nat Rev Mol Cell Biol2001;2:179–87.

8. Neefjes JJ, Momburg F, Hammerling GJ. Selective and ATP-dependenttranslocation of peptides by the MHC-encoded transporter. Science 1993;261:769–71.

9. Fruci D, Niedermann G, Butler RH, van Endert PM. Efficient MHC class I-independent amino-terminal trimming of epitope precursor peptides inthe endoplasmic reticulum. Immunity 2001;15:467–76.

10. Serwold T,Gaw S, Shastri N. ER aminopeptidases generate a unique pool ofpeptides for MHC class I molecules. Nat Immunol 2001;2:644–51.

11. James E, Bailey I, Sugiyarto G, Elliott T. Induction of protective antitumorimmunity through attenuation of ERAAP function. J Immunol 2013;190:5839–46.

12. Textor A, Schmidt K, Kloetzel PM, Weißbrich B, Perez C, Charo J, et al.Preventing tumor escape by targeting a post-proteasomal trimming inde-pendent epitope. J Exp Med 2016;213:2333–2348.

13. Saveanu L, Carroll O, Weimershaus M, Guermonprez P, Firat E, Lindo V,et al. IRAP identifies an endosomal compartment required for MHC class Icross-presentation. Science 2009;325:213–7.

14. Yan J, ParekhVV,Mendez-FernandezY,Olivares-Villag�omezD,Dragovic S,Hill T, et al. In vivo role of ER-associated peptidase activity in tailoringpeptides for presentationbyMHCclass Ia and class Ibmolecules. J ExpMed2006;203:647–59.

15. Firat E, Saveanu L, Aichele P, Staeheli P, Huai J, Gaedicke S, et al. Therole of endoplasmic reticulum-associated aminopeptidase 1 in immu-nity to infection and in cross-presentation. J Immunol 2007;178:2241–8.

16. Willimsky G, Cz�ehM, Loddenkemper C, Gellermann J, Schmidt K, Wust P,et al. Immunogenicity of premalignant lesions is the primary cause ofgeneral cytotoxic T lymphocyte unresponsiveness. J Exp Med 2008;205:1687–700.

17. Willimsky G, Blankenstein T. Sporadic immunogenic tumours avoiddestruction by inducing T-cell tolerance. Nature 2005;437:141–6.

18. York IA, Brehm MA, Zendzian S, Towne CF, Rock KL. Endoplasmicreticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented pep-tides in vivo and plays an important role in immunodominance. Proc NatlAcad Sci U S A 2006;103:9202–7.

ERAP1 Is Crucial for Peripheral T-cell Expansion

www.aacrjournals.org Cancer Res; 78(12) June 15, 2018 3253

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

19. Mylin LM, Schell TD, Roberts D, Epler M, Boesteanu A, Collins EJ, et al.Quantitation of CD8(þ) T-lymphocyte responses to multiple epitopesfrom simian virus 40 (SV40) large T antigen in C57BL/6 mice immunizedwith SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombi-nants expressing full-length T antigen or epitope minigenes. J Virol2000;74:6922–34.

20. Hammer GE, Gonzalez F, Champsaur M, Cado D, Shastri N. Theaminopeptidase ERAAP shapes the peptide repertoire displayed bymajor histocompatibility complex class I molecules. Nat Immunol2006;7:103–12.

21. Cifaldi L, Lo Monaco E, Forloni M, Giorda E, Lorenzi S, Petrini S, et al.Natural killer cells efficiently reject lymphoma silenced for the endoplas-mic reticulum aminopeptidase associated with antigen processing. CancerRes 2011;71:1597–606.

22. Cifaldi L, Romania P, Falco M, Lorenzi S, Meazza R, Petrini S, et al. ERAP1regulates natural killer cell function by controlling the engagement ofinhibitory receptors. Cancer Res 2015;75:824–34.

23. Schmitt TM, Stromnes IM, Chapuis AG, Greenberg PD. Newstrategies in engineering T-cell receptor gene-modified T cells to

more effectively target malignancies. Clin Cancer Res 2015;21:5191–7.

24. Dummer W, Niethammer AG, Baccala R, Lawson BR, Wagner N, ReisfeldRA, et al. T cell homeostatic proliferation elicits effective antitumor auto-immunity. J Clin Invest 2002;110:185–92.

25. Klebanoff CA, Khong HT, Antony PA, Palmer DC, Restifo NP. Sinks,suppressors and antigen presenters: how lymphodepletion enhancesT cell-mediated tumor immunotherapy. Trends Immunol 2005;26:111–7.

26. Reeves E, Colebatch-Bourn A, Elliott T, Edwards CJ, James E. Functionallydistinct ERAP1 allotype combinations distinguish individuals with anky-losing spondylitis. Proc Natl Acad Sci U S A 2014;111:17594–9.

27. Reeves E, Edwards CJ, Elliott T, James E. Naturally occurring ERAP1haplotypes encode functionally distinct alleles with fine substrate speci-ficity. J Immunol 2013;191:35–43.

28. Roberts AR, Appleton LH, Cortes A, Vecellio M, Lau J, Watts L, et al. ERAP1association with ankylosing spondylitis is attributable to common geno-types rather than rare haplotype combinations. Proc Natl Acad Sci U S A2017;114:558–561.

Cancer Res; 78(12) June 15, 2018 Cancer Research3254

Schmidt et al.

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946

2018;78:3243-3254. Published OnlineFirst March 20, 2018.Cancer Res   Karin Schmidt, Christin Keller, Anja A. Kühl, et al.   of Adoptive T-cell Therapy in MiceERAP1-Dependent Antigen Cross-Presentation Determines Efficacy

  Updated version

  10.1158/0008-5472.CAN-17-1946doi:

Access the most recent version of this article at:

  Material

Supplementary

  http://cancerres.aacrjournals.org/content/suppl/2018/03/20/0008-5472.CAN-17-1946.DC1

Access the most recent supplemental material at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/78/12/3243.full#ref-list-1

This article cites 28 articles, 18 of which you can access for free at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.org

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/78/12/3243To request permission to re-use all or part of this article, use this link

on November 2, 2020. © 2018 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-1946