Natural Killer Cells Efficiently Reject Lymphoma Silenced for the … · knockout mice with cells from wild-type mice, orvice versa, results in potent CD8þ T-cell responses (11,

Microenvironment and Immunology

Natural Killer Cells Efficiently Reject Lymphoma Silenced forthe Endoplasmic Reticulum Aminopeptidase Associatedwith Antigen Processing

Loredana Cifaldi1, Elisa Lo Monaco2, Matteo Forloni1, Ezio Giorda1, Silvia Lorenzi1, Stefania Petrini1,Elisa Tremante2, Daniela Pende3, Franco Locatelli1,4, Patrizio Giacomini2, and Doriana Fruci1

AbstractThe endoplasmic reticulum aminopeptidase ERAAP is involved in the final trimming of peptides for

presentation by MHC class I (MHC-I) molecules. Herein, we show that ERAAP silencing results in MHC-Ipeptide-loading defects eliciting rejection of the murine T-cell lymphoma RMA in syngeneic mice. Although CD4and CD8 T cells are also involved, rejection is mainly due to an immediate natural killer (NK) cell response anddepends on the MHC-I-peptide repertoire because replacement of endogenous peptides with correctly trimmed,high-affinity peptides is sufficient to restore an NK-protective effect of MHC-I molecules through the Ly49C/I NKinhibitory receptors. At the crossroad between innate and adaptive immunity, ERAAP is therefore unique in itstwo-tiered ability to control tumor immunogenicity. Because a large fraction of human tumors express highlevels of the homologous ERAP1 and/or ERAP2, the present findings highlight a convenient, novel target forcancer immunotherapy. Cancer Res; 71(5); 1597–606. �2011 AACR.

Introduction

Antigenic peptides are generated in the cytoplasm asproteolytic intermediates by degradation of endogenous pro-teins through the multicatalytic proteasome and other pro-teases (1). Proteolytic intermediates are then translocated intothe endoplasmic reticulum (ER) by the transporter associatedwith antigen processing (TAP) and further processed by ERaminopeptidases, that is ERAAP in mice (2) and ERAP1 andERAP2 in humans (3–5), before being loaded onto MHC class I(MHC-I) molecules.Inhibition or loss of cytoplasmic proteases, the TAP

transporter and components of the peptide-loading complexresults in the marked downregulation of MHC-I expressionon the cell surface (6–10). In contrast, inhibition of ERaminopeptidases by siRNA or in ERAAP-knockout micecauses a partial suppression not exceeding 60% on MHC-Isurface expression (2, 5). Yet, immunization of ERAAP-

knockout mice with cells from wild-type mice, or vice versa,results in potent CD8þ T-cell responses (11, 12), suggestingqualitative and quantitative alterations in the MHC-I-pep-tide (pMHC) repertoire. Consistent with these findings, massspectrometric analysis revealed a marked increases in thelength of endogenous peptides presented by MHC-I in micelacking ERAAP (13), but whether or not ERAAP-dependentpMHC alterations affect innate immune responses is pre-sently unknown.

In agreement with these studies, we have previouslyobserved that an altered expression of ERAP1 and ERAP2in human tumors as compared with their normal counterparts(14) results in an abnormal MHC-I expression (15), but howthis might affect host immune responses against tumors(either adaptive or innate) is presently unknown.

To address this issue, ERAAP expression was stably reducedin the murine T-cell lymphoma RMA by transfection ofERAAP-targeted microRNA. Taking advantage of in vivo andex vivo molecular and functional studies, we show herein thatERAAP-silenced RMA cells are readily rejected by syngeneicmice mainly through a tumor-specific natural killer (NK) cellresponse, due to poor pMHC-I engagement of Ly49C/I NKinhibitory receptors.

Materials and Methods

Mice and cell linesSix- to 8-week-old inbred female C57BL/6 (Charles Rivers

Laboratories) and C57BL/6 nude (Taconic) mice were main-tained at the Animal Resource Facilities in accordancewith theexperimental ethics committee guidelines. RMA (16), RMA-S(16–19), and YAC-1 (20) T-lymphoma cell lines (courtesy of R.

Authors' Affiliations: 1Oncohaematology Department, IRCCS, OspedalePediatrico Bambino Gesù; 2Laboratory of Immunology CRS, Regina ElenaCancer Institute, Rome, Italy; 3Istituto Nazionale per la Ricerca sul Cancro,Genoa, Italy; and 4University of Pavia, Pavia, Italy

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

E. Lo Monaco and M. Forloni contributed equally to this work.

Corresponding Author: Doriana Fruci, Laboratorio di Immunoncologia,Dipartimento di Oncoematologia Pediatrica, IRCCS, Ospedale PediatricoBambino Gesù, Piazza Sant'Onofrio 4, 00165 Rome, Italy. Phone: 39-06-68592657; Fax: 39-06-68592904; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-3326

�2011 American Association for Cancer Research.

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Kiessling and A. Santoni) were authenticated by staining withH-2 allele–specific monoclonal antibodies (mAb).

RNA interferenceMicro RNAs (miRNA) to mouse ERAAP (NM_030711) were

cloned into the pcDNA6.2-GW/EmGFP-miR vector, trans-fected into RMA cells by electroporation (21), selected inblasticidin (Invitrogen), sorted for green fluorescent protein(GFP) expression, and cloned. For further details see Supple-mentary Material.

Antibodies and peptidesmAbs to H-2Kb and H-2Db (K204), H-2Kb (Y-3), H-2Db

(B22.249), NK1.1 (PK136), CD4 (GK1.5), and CD8 (53-6-72)were used. Peptides SIINFEKL (22), ASNENMETM (23), andSLYNTVATL (24) and SIINFEAL (25) were used. A detailedlist of commercial fluorescent antibodies for flow cytometryand peptide features used is provided in SupplementaryMaterials.

Flow cytometry, immunofluorescence, proteinbiochemistry, and aminopeptidase activity

Flow cytometry was done on FASCalibur and FASCantoII(BD-PharMingen). Acetone-fixed tumor sections were stainedwith specific mAbs and analyzed by confocal microscope.Western blotting, microsomal aminopeptidase activity tests,pulse-chase, and in vitro assemblywere performed as described(15, 26, 27). For further details see Supplementary Materials.

Surface stabilityThe rate of generation of newly assembled pMHC-I

complexes, the dissociation of preformed pMHC-I complexes,and the replacement of the endogenous MHC-I–binding pep-tides were detected as described (11, 25, 28). To detect theOVA257–264 peptide replacement, cells were stained with25-D1.16 mAb (29).

In vivo tumor challenge, immunodepletion, andperitoneal clearance assay

Tumor cells (104) were injected subcutaneously into theright flank of 6- to 8-week-old syngeneic C57BL/6 and C57BL/6nude mice. Tumor growth was monitored every 2 days. Forethical reasons, animals were sacrificed when tumor reached avolume greater than 5 cm3. NK1.1þ, CD4þ, and CD8þ cellswere depleted in vivo by intraperitoneal administration of200 mg of PK136, GK1.5, or 53.6-72 at day �1, 0, 1, 3, 7, and 15relative to tumor inoculation (30). Alternatively, tumor cells(106) were injected intraperitoneally. Apoptosis of tumor(GFPþ) cells was evaluated with APC-AnnexinV (BD-PharMin-gen) and propidium iodide (PI; Sigma-Aldrich), whereas peri-toneal exudate (PE) leukocytes (GFP�) were stained withspecific antibodies. For further details see SupplementaryMaterials.

Interleukin 2–activated splenocytesSingle-cell suspensions fromC57BL/6 spleens were cultured

(15 � 106 cells/mL) with 100 ng/mL recombinant human

Figure 1. Silencing of ERAAPaffects MHC-I surface expressionin RMA cells. A, immunoblotanalysis of ERAAP expression inRMA (�), RMA-scramble (sc), andrepresentative RMA-siERAAP cellclones. An ERp57 Ab (14) wasused for normalization. B, kineticsof Leu-AMC degradation(fluorescence units) inmicrosomes from RMA pretreatedat 37�C for 1 hour with either 0.5mmol/L DTT (DTT) or 90 mmol/L/0.5 mmol/L Leu-SH/DTT (Leu-SH)and in microsomes from untreatedRMA, RMA-sc, and RMA-siERAAP clones 5, 6, and 22.Statistically significant differences(RMA-sc vs. RMA-siERAAPclones and DTT-treated vs. Leu-SH–treated RMA) are indicated(*, P < 0.0001). C, flow cytometricanalysis of MHC-I expression inRMA pretreated 18 hours at 37�Cwith either 0.5 mmol/L DTT or90 mmol/L/0.5 mmol/L Leu-SH orelse left untreated (�) and RMA-Scells (top) and MHC-I, CD5, andCD45 expression in RMA, RMA-sc, and RMA siERAAP clones 5, 6,and 22 (bottom). Isotype-matchedcontrol antibody staining did notexceed an MFI (meanfluorescence intensity) value of 5.

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interleukin 2 (IL-2; PeproTech) for 7 days. The cells weresorted on FACSAria by gating on NK1.1þCD3�Ly49C/Iþ andNK1.1þCD3�Ly49C/I� and then cultured (2 � 106/mL) for 40hours with IL-2 and used as cytotoxicity effectors (25).

NK functional assaysNK cell cytotoxic activity was evaluated by a standard 5-

hour 51Cr release assay (31). The degranulation assay wasperformed by coculturing target and effector cells at a 1:1 ratiofor 6 hours in the presence of GolgiStop (BD) and anti-CD107a(32–35), as described in detail in Supplementary Materials.

Statistical analysisStatistical significance was assessed by the 2-tailed

unpaired Student's t test. Survival data are presented asKaplan–Meyer plots.

Results

Downregulation of ERAAP affects MHC-I surfaceexpression in RMA cellsTo assess the relevance of ER peptide trimming on tumor-

igenicity, we stably suppressed the expression of ERAAP in the

murine T-cell lymphoma RMA of C57BL/6 origin by transfec-tion of vectors encoding GFP in combination with microRNAsto either ERAAP (RMA-siERAAP) or a control, scrambledsequence (RMA-scramble). ERAAP mRNA levels were similarin parental cells and 10 RMA-scramble clones but weredecreased, although to a different extent, in 31 distinctRMA-siERAAP clones (Supplementary Fig. S1). Eight repre-sentative RMA-siERAAP clones and 1 RMA-scramble cloneselected at random (hitherto RMA-sc) were selected andtested by Western blotting to confirm specific ERAAP down-regulation at the protein level. RMA-siERAAP clone 22 (RMA-siERAAP-22) showed the lowest ERAAP levels, correspondingto a relative decrease of 90% compared with the parental RMAcells and RMA-sc, whereas RMA-siERAAP clones 5, 6, 28, and50 showed a less pronounced downregulation (Fig. 1A). Ami-nopeptidase activity in isolated microsomes from 3 represen-tative RMA-siERAAP clones and control cells, RMA-sc andRMA cells, was proportional to the amount of the aminopep-tidase expressed, whereas essentially no substrate cleavagewas seen in microsomes from RMA cells treated with Leu-SH,a specific aminopeptidase inhibitor (Fig. 1B). MHC-I surfaceexpression was decreased in these RMA-siERAAP clones by48.5% to 56%, as assessed by K204 mAb, whereas no change in

Figure 2. Silencing of ERAAP affects MHC-I stability. A, cells were pulse-labeled with 35S-methionine for 20 minutes and chased as indicated. H-2Kb

molecules were immunoprecipitated with Y-3 mAb, digested with Endo H (þ) or mock-incubated (�), and resolved by SDS-PAGE. Endo H–resistant (r)and Endo H–sensitive (s) HC bands are shown. B, cells were metabolically labeled with 35S-methionine for 2 hours, lysed, and either mock-incubated (�) orincubated 3 hours with 50 mg/mL of SIINFEKL (þ) at 4�C and 37�C. Immunoprecipitates were analyzed as above. C, following dissociation and recoveryof preexisting MHC-I complexes (see the text), cells were stained with K204 mAb and analyzed by flow cytometry. Results are presented as recovery ofMFI at different time points. Statistically significant differences (RMA-sc vs. RMA-siERAAP) are indicated (*, P < 0.05; **, P < 0.01). D, cells were culturedin 10 mmol/L brefeldin A as indicated, stained with K204 mAb, and analyzed as in (C).

ERAAP Regulates NK Cell–Mediated Tumor Rejection

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the expression of control surface markers CD5 and CD45 wasdetected (Fig. 1C). MHC-I expression was similarly reduced(52%) in RMA cells treated with Leu-SH (Fig. 1C). Similarresults were obtained using Y3 and B22.249 antibodies specificto H-2Kb and H-2Db, respectively (Supplementary Table SI).

Thus, downregulation of ERAAP affects both microsomalaminopeptidase activity and MHC-I surface expression inRMA cells.

Instability of pMHC-I complexes in RMA-siERAAP cellsERAAP deficiency has been associated with alterations in

MHC-I assembly and/or stability (11, 36). To verify whetherreduced MHC-I surface expression in RMA-siERAAP clonesis a consequence of the limited generation of stable pMHC-Icomplexes, the biochemical features of MHC-I expressed byRMA-siERAAP-22 cells were compared with those of RMA,RMA-sc, and TAP-defective RMA-S cells (37). In pulse-chaseexperiments (Fig. 2A), the conformational antibody Y-3 (37,38) detected 20% less Kb in RMA-siERAAP-22 than in RMAand RMA-sc cells at the 00 chase point, and a further 20%reduction was evident at the 2-hour point despite completematuration of endoglycosidase Endo H–sensitive (s) intoEndo H–resistant (r) Kb glycoforms (Fig. 2A). Much lower Kb

levels and more than 85% decrease in the mature glycancomponent were instead detected in RMA-S cells, asexpected (37, 38).

To investigate heavy chain (HC) stability and associationwith b2m, detergent-soluble H2-Kb molecules were tested byin vitro assembly in the absence and presence of a large excessof the high-affinity H-2Kb–binding peptide SIINFEKL at 2different temperatures (Fig. 2B). Lesser amounts of Kb andassociated b2m were detected in RMA-siERAAP cells than inparental RMA and RMA-sc cells, although these 3 cells weremuch more similar to one another than to deeply defectiveRMA-S cells, which was confirmed (38) to be composed of lowlevels of thermally unstable, mostly immature, Kb glycoforms(Fig. 2B). Similar results were obtained using K204 mAb(Supplementary Fig. S2A). Thus, ERAAP silencing affectsslightly, if at all, thermal stability and peptide receptivity invitro but detectably impairs the spontaneous assembly andintracellular transport of Kb in vivo.

To further document MHC-I instability in RMA-siERAAPclones, the generation of newly assembled pMHC-I complexesand the dissociation of preformed pMHC-I complexes wereevaluated in RMA-siERAAP clones. In the former approach,pMHC-I complexes were denatured on the cell surface withisotonic, low pH buffer and then allowed to regenerate byreturning the cells to culture conditions (37), whereas in thelatter approach, the surface expression of newly assembledMHC-I molecules was inhibited by treatment with brefeldin A.The reconstitution of MHC-I surface expression (monitoredover time by flow cytometry with K204 mAb) was slightly

Figure 3. Downregulation ofERAAP renders mice resistant totumor challenge. A, C57BL/6 mice(n ¼ 10 per group) werechallenged by subcutaneousinoculation of 104 RMA, RMA-S,RMA-sc, or RMA-siERAAP-22cells. Tumor growth (mean � SD)was monitored every 2 days.Statistically significant differencesare indicated (*, P ¼ 0.007).B, overall survival (Kaplan–Meier).Results are representative of 4independent experiments.C, frozen tumor sections frommice sacrificed 12 days afterchallenge with RMA-sc and RMA-siERAAP-22 were stained withAPC-NK1.1 or PECy5-CD8,nuclear counterstained withHoechst 33342, and analyzed byconfocal microscopy (left). CD8þ

and NK1.1þ cells (percentage �SD of infiltrating lymphocytes)were averaged in 3 microscopicfields (right). Scale bar, 40 mm.

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slower in RMA-siERAAP cell clones 5 and 22 than in RMA andRMA-sc cells (Fig. 2C). After 5 hours in culture, control cellsrecovered 80% of the MHC-I surface expression, whereasRMA-siERAAP cell clones 5 and 22 recovered only 40%(Fig. 2C and Supplementary Fig. S2B). During the same period,the MHC-I dissociation rate was faster in RMA-siERAAPclones 5 and 22 than in RMA and RMA-sc cells (Fig. 2D),suggesting that a greater fraction of surface MHC-I in RMA-siERAAP cells is unstable.Altogether, these results demonstrate that the downregula-

tion of surface MHC-I expression in RMA-siERAAP cells is dueto both a stoichiometric loss of MHC-I molecules since earlybiosynthetic stages, and the instability of pMHC-I complexeson the cell surface.

In vivo evaluation of host immune responses againstRMA-siERAAP cellsTo investigate whether ERAAP deficiency affects RMA

tumorigenicity, we subcutaneously inoculated RMA-siER-

AAP-22, RMA-sc, RMA, and RMA-S cells in syngeneicC57BL/6 mice. All mice injected with control RMA andRMA-sc cells developed progressively enlarging tumors anddied within 32 days, whereas mice injected with RMA-siER-AAP-22 and RMA-S did not develop palpable tumors in mostcases (Fig. 3A and B). Only 3 of 10 mice injected with RMA-siERAAP-22 and 2 of 10 mice injected with RMA-S developedpalpable tumors within 15 days after inoculation (Fig. 3A andB). Of these, one RMA-siERAAP-22 and both RMA-S tumorscompletely regressed in a few days whereas the remaining 2RMA-siERAAP-22 tumors grew very slowly and did not killedtheir hosts by day 50.

Rejection of RMA-siERAAP-22 is likely mediated by a hostimmune response because the in vitro proliferation rates andapoptotic state of RMA-siERAAP-22, RMA, and RMA-sc cellswere very similar (Supplementary Fig. S3A and B). In line withthis interpretation, confocal microscopy revealed a significantincrease of tumor-infiltrating NK1.1þ and CD8þ cells in RMA-siERAAP-22 tumors as compared with RMA-sc tumors

Figure 4. Tumor rejection inducedby ERAAP downregulationrequires intact NK1.1þ, CD4þ, andCD8þ cell populations. Mice(n ¼ 10 per group) wereimmunodepleted byintraperitoneal injection with theindicated mAbs and challengedsubcutaneously with 104 RMA-siERAAP-22 cells. Tumor growth(A, C) and Kaplan–Meyer survivalcurves (B, D) of single (A, B) andcombined (C, D) depletion in 1 of 2representative experiments.Statistically significant differencesare indicated (*, P < 0.05; **,P < 0.000001).

Figure 5. CD107a induction and IFN-g production by NK cells in response to RMA-siERAAP cells. Flow cytometric analysis of CD107a expressionand IFN-g production by splenic NK1.1þCD3� cells from poly(I:C)-treated C57BL/6 mice cocultured with the indicated target cells. Numbers indicatepercentages of single- and double-positive cells.






(Fig. 3C). The increased number of CD8þ and NK1.1þ lym-phocytes in RMA-siERAAP-22 tumors was further confirmedby flow cytometry of cell suspensions from tumor lesions(Supplementary Fig. S3C).

Altogether, these data indicate an effective host immuneresponse against ERAAP downregulated RMA cells.

Rejection of RMA-siERAAP cells requires intact NK cellsand CD8 and CD4 T-cell responses

To identify the immune effectors rejecting RMA-siERAAP-22 tumors, C57BL/6 mice were antibody depleted of CD4þ,CD8þ, and NK1.1þ cell populations and challenged by sub-cutaneous injection with RMA-siERAAP-22. Depletion of the

Figure 6. Cytotoxic activity of NK cells from mice intraperitoneally challenged with RMA-siERAAP clone 22. C57BL/6 mice, treated with an irrelevantIgG or were NK1.1 depleted, were injected intraperitoneally with 106 RMA-sc and/or RMA-siERAAP-22 cells, respectively. Unfractionated PE cells wererecovered from the peritoneal cavity at the indicated times and analyzed by either flow cytometry (A–D) or cytotoxicity (E). A, marked gates correspond tothe percentage of GFPþ tumor cells. B, apoptotic state of GFPþ tumor cells stained with AnnexinV and PI. The percentage of cells in each quadrant is indicated.C, lymphocyte subsets were enumerated on mAb staining. Statistically significant differences (RMA-sc vs. RMA-siERAAP-22) are indicated (*, P < 0.05).D, unfractionated PE cells collected at the indicated times from mice inoculated with saline, RMA-sc, or RMA-siERAAP-22 were tested as effectors in astandard 51Cr release assay on the indicated targets at different effector/target (E:T) ratios. E, CD107a expression of NK1.1þCD3� PE cells from miceinoculated with tumor cells as in (D) in response to YAC-1, RMA-S, RMA-sc, or RMA-siERAAP-22 target cells. Data are representative of 3 independentexperiments.

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different lymphocyte subset restored the growth of RMA-siERAAP-22 during the 17-day observation period but onlypartially and to a different extent (Fig. 4A). The differentcontribution of each lymphocyte subset was more evidentin the 40-day Kaplan–Meyer curve (Fig. 4B). Similar to micetreated with an irrelevant IgG and challenged with controlRMA-sc cells, depletion of NK1.1þ cells resulted in palpabletumors by day 15 (Fig. 4A) that rapidly grew causing death ofall mice by day 30 (Fig. 4B). In contrast, depletion of eitherCD4þ or CD8þ T cells had much slighter effects, resulting ina slower tumor growth (Fig. 4A) and death of 40% and 20%of the mice, respectively, 5 weeks after challenge (Fig. 4B).Remarkably, combined NK1.1þ/CD4þ depletion, with or with-out CD8þ depletion, as well as NK1.1þ/CD8þ depletion,completely restored RMA-siERAAP-22 tumor growth toRMA-sc levels. In contrast, the effect of double CD4þ/CD8þ

depletion was marginal during the first 17 days (Fig. 4C) andresulted in delayed tumor growth and death of 60% of mice29 days after challenge (Fig. 4D). Treatment with an irrelevantIgG did not affect tumor rejection (Fig. 4). Finally, C57BL/6nude mice efficiently rejected RMA-siERAAP-22 and RMA-Scells as immunocompetent mice, emphasizing the minorcontribution of CD4þ and CD8þ T cells to tumor control(Supplementary Fig. S4).Thus, rejection of RMA-siERAAPmainly depends on NK1.1þ

cells, but complete tumor control is sustained by a synergybetween innate (NK1.1) and adaptive (CD4 and CD8) immunesubsets.

Specific recognition of RMA-siERAAP cells by NK cellsTo demonstrate specific recognition by NK cells, RMA-

siERAAP-22 cells were compared to the well-known NK-sus-ceptible targets YAC-1 and RMA-S in their ability to induce theexpression of CD107a (32–35), a marker of granule exocytosis,and the production of IFN-g in splenic NK cells. Remarkably(Fig. 5), NK cell activation by RMA-siERAAP-22, YAC-1, andRMA-S was similar and much greater than that by RMA andRMA-sc cells.Next, RMA-siERAAP-22 cell targets and NK cell effectors

were studied as single-cell suspensions in a model of intra-peritoneal tumor growth (39, 40). RMA-siERAAP-22 cells andRMA-sc cells were injected intraperitoneally into C57BL/6mice, either treated with an irrelevant IgG or were NK1.1depleted. PE cells were harvested at different times andanalyzed by flow cytometry for the presence of GFPþ tumorcells and GFP� lymphocyte subsets (Fig. 6 and SupplementaryFig. S5).Similar to subcutaneous injection experiments (Figs. 3 and

4), RMA-siERAAP-22 cells were rejected, but they grew inNK1.1-depleted mice as RMA-sc cells grow in control mice(Fig. 6A). Interestingly, GFPþ RMA-siERAAP-22 cells began todecrease at 12 hours in the peritoneal cavity and disappearedalmost completely at 48 hours (Fig. 6A). Their staining withAnnexinV and PI revealed very rapid killing kinetics, earlyapoptotic cells (AnnexinVþ/PI�) appearing at 4 hours or ear-lier, and progressively turning into late apoptotic cells(AnnexinVþ/PIþ) at later times (Fig. 6B). Remarkably, apop-totic death was negligible when the same RMA-siERAAP-22

cells were injected into NK1.1þ-depleted mice or RMA-sc cellswere injected into C57BL/6 mice (Fig. 6B). The role of NK cellsas mediators of this apoptotic death was strongly supported bythe increased number of NK1.1þ/CD3� cells in PE from miceinoculated with RMA-siERAAP-22 at 4 hours (Fig. 6C), that is,coinciding with the beginning of apoptotic decline. Likewise,the virtual absence of NK1.1þ/CD3þ PE cells ruled out anunlikely involvement of NK T cells in tumor rejection (Fig. 6C).

These results conclusively document the direct participa-tion andmajor role of NK cells at early stages of RMA-siERAAPtumor control and demonstrate that 12 hours following tumorchallenge are sufficient to provide PE cells with the ability tokill RMA-siERAAP cells.

Ex vivo evaluation of NK immune responses induced byRMA-siERAAP cells

To characterize NK cells infiltrated into the peritoneal cavityupon inoculation of tumor cells, PE cells frommice inoculated

Figure 7. Peptide-specific protection of RMA-siERAAP cells againstNK-mediated cytotoxicity. RMA-sc and RMA-siERAAP-22 cells wereincubated in medium containing 2% fetal calf serum (FCS) and theindicated peptides (50 mmol/L) at 26�C for 18 hours and then (A) incubatedfor additional 5 hours at 37�C in medium containing 10% FCS andpeptides and analyzed by flow cytometry following staining with mAbsK204 and 25-D1.16, or (B and C) labeled with 51Cr and tested in thepresence of peptides for sensitivity to lysis by (B) PE cells from miceinjected intraperitoneally with RMA-siERAAP-22, or (C)NK1.1þCD3�Ly49C/Iþ and NK1.1þCD3�Ly49C/I� effector cells sortedfrom IL-2–activated splenocytes. Data are representative of 3 independentexperiments (� SD of triplicate assays).






with RMA-siERAAP-22 cells, RMA-sc cells, or saline at differenttimes were used as effectors on YAC-1, RMA-S, RMA-siERAAP-22, and RMA-sc cells in a standard 51Cr release assay (ref. 39;Fig. 6D). PE cells from mice inoculated with saline were pooreffectors and failed to reveal any difference in NK susceptibilityamong YAC-1, RMA-S, RMA-sc, and RMA-siERAAP-22. Incontrast, PE cells from mice inoculated with either tumorwere much better effectors for all the 4 targets, particularlyfor YAC-1 and RMA-siERAAP-22 after 4 and 12 hours frominjection, respectively. This increased lytic activity was furtherconfirmed by a degranulation assay (Fig. 6E).

Next, we assayed the ability of PE cells frommice inoculatedwith RMA-siERAAP-22 to kill RMA-siERAAP-22 in which theendogenous MHC-I peptides were exchanged by incubationwith a large excess of the high-affinity peptides ASNENMETM(23) and SIINFEKL (22). As expected, the MHC-I–specific K204mAb did not detect any significant difference regardless of thepeptide added. In contrast, 25-D1.16mAb (29) specific to theH-2Kb/SIINFEKL complex revealed an efficient SIINFEKLpeptideexchange on the cell surface (Fig. 7A). Most interestingly, bothpeptides, but not the irrelevant SLYNTVATL ligand, totallyprotected RMA-siERAAP-22 from PE cell–mediated lysis(Fig. 7B). Thus, PE cells preferentially kill RMA-siERAAP-22.Moreover, replacement of preexisting endogenous peptides isnecessary and sufficient to restore the NK-protective effect ofMHC-I molecules, suggesting that MHC-I misfolding entirelydepends on the peptide pool of RMA-siERAAP cells.

RMA-siERAAP cells are lysed by NK cells, due toimproper recognition of pMHC-I complexes by Ly49C/I

To identify theNKcell receptor(s) involved in the rejection ofRMA-siERAAP cells, we focused our attention on Ly49C/I, themain MHC-I–monitoring inhibitory receptors expressed in H-2b mice (41). Ly49C/I receptor engagement has been shown todepend on the composition ofMHC-I–bound peptides (25, 42–44). RMA-siERAAP and RMA-sc cells were tested as targets ofNK1.1þCD3�Ly49C/Iþ and NK1.1þCD3�Ly49C/I� effectorcells isolated from IL-2–activated splenocytes of C57BL/6mice.NK1.1þCD3�Ly49C/Iþ effector cells efficiently killed RMA-siERAAP-22 cells but not RMA-sc cells (Fig. 7C). Interestingly,as for PE-mediated lysis (Fig. 7B), loading with SIINFEKLpeptide, but not with the irrelevant SLYNTVATL ligand, totallyprotected RMA-siERAAP-22 from lysis (Fig. 7C). As expected,loading with the K7A-substituted OVA-derived peptide SIIN-FEAL, known to support Kb assembly but contribute poorRMA-S protection from NK lysis (25), only partially protectedRMA-siERAAP-22 (Fig. 7C). As previously described for RMAand RMA-S cells (25), NK1.1þCD3�Ly49C/I� effector cellskilled RMA-sc and RMA-siERAAP target cells with similarefficiency.

Thus, NK cells lyse RMA-siERAAP cells because pMHC-Icomplexes fail to engage the inhibitory Ly49C/I receptors.

Discussion

Herein, ERAAP downregulation in RMA lymphoma cellswas shown to induce NK cell–mediated, and subsequently T-cell–mediated (CD4 and CD8), rejection in syngeneic mice. To

our knowledge, this is the first demonstration that ERAAPregulates tumorigenicity. CD8 involvement might have beenanticipated from previous studies, because in ERAAP-knock-out mice a marked alteration in the pMHC-I repertoire (11, 12)resulted in potent CD8 T-cell responses. In contrast, a majorNK cell triggering effect was particularly surprising and totallyunanticipated, because ERAAP downregulation caused amod-est (about 50%) suppression of MHC-I expression in all studies(2, 11, 12, 36) including the present one.

Our RMA-siERAAP clones display alterations in MHC-Isurface expression and surface turnover similar to thoseseen in knockout mice (11, 36). In addition, these clonesshow a progressive stoichiometric loss of MHC-I molecules,evident since early biosynthetic stages, resulting in areduced accumulation of b2m-associated, conformed heavychains. These alterations are much slighter than those seenin TAP-defective RMA-S cells (16), and yet they indirectlydocument a subtle conformational change that appears tobe sufficient to completely derange MHC-I interactions withthe prominent NK receptor and MHC-I folding sensorLy49C/I. This effect is not due to a simple quantitativereduction in surface MHC-I, as in vitro assembly and in vivopulse with canonical, trimmed peptides rescued peptide-specific conformational epitopes, as well as receptor engage-ment. It is also highly unlikely to result from a directinterference of an altered pMHC-I peptide pool with Ly49binding, because the crystallographic Ly49C footprint on Kb

molecules involves a broad cavity beneath the floor of thepeptide-binding groove opposite to, and not including,bound peptides (44). To explain how different peptidesmight differently affect receptor engagement, it has beensuggested that peptide-selective conformational changes aretransmitted from the peptide-filled groove to the H-2Kb/Ly49C interface through a "domino" effect involving succes-sive positional adjustments of not only heavy chain but alsob2m residues (25, 41). Altogether, in the context of thealterations to the pMHC-I pool previously described inERAAP knockouts (11), the presently described decreasesin MHC-I stability, b2m association, and intracellular trans-port, as well as the peptide-reversible susceptibility to NKcell lysis of RMA-siERAAP cells, are fully consistent withcrystallographic models of peptide-driven/conformation-dependent Ly49C engagement.

In summary, and quite strikingly, MHC-I molecules synthe-sized in cells impaired in N-terminal peptide trimming havebeen shown by others to be sufficiently conformed to presentantigen to CD8 T cells (12) and by us to be insufficientlyconformed to inhibit NK cells. Our immune subset depletionexperiments provide in vivo evidence for both mechanisms,NK cell killing of tumor cells being clearly preponderant andoccurring at an earlier stage than CD4-mediated and CD8-mediated rejection. Indeed, by inciting recognition of missingself and altered self at the same time, ERAAP appears toregulate tumor immunogenicity at a crucial crossroadbetween innate and adaptive immunity.

In the context of this synergistic effect, NK cells are clearlythe major players not only because they contribute the majorfraction of the phenotypically observed tumor control but also

Cifaldi et al.





because the immediate burst of NK-mediated apoptotic deathleading to massive tumor clearance in vivo begins just 4 hoursfollowing tumor challenge, for example, at a time that isinconsistent with any adaptive response. On the other hand,our in vivo experiments show that depletion of CD4 and CD8subsets results in late tumor growth, which in most instancesdoes not kill mice for up to 50 days. This favors the possibilitythat CD4 and CD8 effectors are involved in the control ofRMA-siERAAP tumors that have escaped NK attention and isreminiscent of the equilibrium phase of immune editingmodels of tumor immune surveillance (45).In humans, we have previously shown that most tumor cells

(15) and neoplastic tissues (14) express from intermediate tohigh levels of ERAP1 and ERAP2, tumor cell lines falling in thelowest 10% range of expression for either aminopeptidasebeing a minority. In light of the dramatic enhancement on NKcell lysis presently observed by reducing ERAAP expression toa residual 10%, it is tempting to speculate that ERAP-lowtumor phenotypes are subjected to immune counterselectionin human patients. It may be of interest to determine whetherin humans, as in mice, such a low expression in tumor lesionsis associated with increased NK cell and T-cell infiltrates and afavorable outcome due to tumor cell killing by both innate andadaptive immunity.Suppression of peptide trimming, using small drugs speci-

fically inhibiting ERAAP activity, may be easier than interfer-

ing with peptide transport by TAP. Pharmaceuticalsmimicking the functional consequences of ERAAP interfer-ence might unveil novel NK-mediated immunotherapeuticapproaches for treatment of cancer.

Disclosure of Potential Conflicts of Interest

The authors have declared that no conflict of interest exists.

Acknowledgments

The authors thank Drs. N. Shastri and P. van Endert for ERAAP mAb, Dr. R.Kiessling for RMA and RMA-S, Drs. G. Bernardini, C. Cerboni, F. Di Rosa, and A.Santoni for the PK136, GK1.5, and 53-6-72 mAbs, Dr. A. Achour for the K7A-substituted OVA-derived peptide, Drs. P. van Endert, N. Tanigaki, and M.D’Amato for helpful suggestions and critical reading of the manuscript, R.Fraioli and G. Cortese for technical assistance, and Dr. G. Citro for mice facilitysupervision.

Grant Support

Associazione Italiana per la Ricerca sul Cancro (AIRC, Milan, Italy) IG grantto D. Fruci and the special project 5 � 1,000 to F. Locatelli; Italian Ministry ofHealth (Rome, Italy) to P. Giacomini.

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

Received September 10, 2010; revised November 23, 2010; acceptedDecember 15, 2010; published OnlineFirst January 20, 2011.

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2011;71:1597-1606. Published OnlineFirst January 20, 2011.Cancer Res Loredana Cifaldi, Elisa Lo Monaco, Matteo Forloni, et al. Antigen Processing

withthe Endoplasmic Reticulum Aminopeptidase Associated Natural Killer Cells Efficiently Reject Lymphoma Silenced for

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