BPTF Depletion Enhances T-cell Mediated Antitumor Immunity...cocultured with 1 610 splenocytes from na€ve mice and stim-ulated with 1 mg/mL Gp100 25–33 (KVPRNQDWL), TRP2 180-188

Microenvironment and Immunology

BPTF Depletion Enhances T-cell–MediatedAntitumor ImmunityKimberly Mayes1, Suehyb G. Alkhatib1, Kristen Peterson1, Aiman Alhazmi1, Carolyn Song1,Vivian Chan1, Tana Blevins2, Mark Roberts1, Catherine I. Dumur2, Xiang-Yang Wang1, andJoseph W. Landry1

Abstract

Genetic studies in fruit flies have implicated the chromatinremodeling complex nucleosome remodeling factor (NURF) inimmunity, but it has yet to be studied in mammals. Here weshow that its targeting in mice enhances antitumor immunity intwo syngeneic models of cancer. NURF was disabled by silenc-ing of bromodomain PHD-finger containing transcription fac-tor (BPTF), the largest and essential subunit of NURF. We foundthat both CD8þ and CD4þ T cells were necessary for enhancedantitumor activity, with elevated numbers of activated CD8þ Tcells observed in BPTF-deficient tumors. Enhanced cytolyticactivity was observed for CD8þ T cells cocultured with BPTF-silenced cells. Similar effects were not produced with T-cellreceptor transgenic CD8þ T cells, implicating the involvement

of novel antigens. Accordingly, enhanced activity was observedfor individual CD8þ T-cell clones from mice bearing BPTF-silenced tumors. Mechanistic investigations revealed that NURFdirectly regulated the expression of genes encoding immuno-proteasome subunits Psmb8 and Psmb9 and the antigen trans-porter genes Tap1 and Tap2. The PSMB8 inhibitor ONX-0914reversed the effects of BPTF ablation, consistent with a criticalrole for the immunoproteasome in improving tumor immu-nogenicity. Thus, NURF normally suppresses tumor antigenicityand its depletion improves antigen processing, CD8 T-cellcytotoxicity, and antitumor immunity, identifying NURF as acandidate therapeutic target to enhance antitumor immunity.Cancer Res; 76(21); 6183–92. �2016 AACR.

IntroductionTumors that present in the clinic adapt to suppress antitumor

immunity by reducing antigenicity, deregulating immunecheckpoints, or amplifying immune-suppressing cells (1).Immunotherapies block these adaptations and reestablish anti-tumor immunity for therapeutic benefit. In rare cases, immu-notherapies have positive therapeutic outcomes, but in a major-ity of cases they do not have lasting antitumor effects due totumor adaptation (2). There is hope that combination therapymay be more effective, but to achieve these outcomes, we mustdiscover novel pathways and targets that can be exploited fortherapeutic advantage.

Decades of research in epigenetics has characterized manyprotein complexes with important roles in regulating nuclearprocesses. These complexes largely operate on the nucleosome,which is the fundamental unit of chromatin structure (3). Oneclass of complexes that modifies the position and structure ofnucleosomes are theATP-dependent chromatin-remodeling com-plexes (4). These chromatin-remodeling processes are frequently

essential for development, but are not generally necessary for cellsurvival (5–7). In addition to roles in normal development,nucleosome-remodeling complexes have prominent roles in can-cer-specific biology, including putative driver functions for avariety of human cancers (8).

One largely uncharacterized chromatin-remodeling complex isthe nucleosome-remodeling factor (NURF; ref. 9). MammalianNURF is composed of three subunits that include the largest,essential, and unique subunit bromodomain PHD-finger con-taining transcription factor (BPTF), the ATPase SNF2L, and a WDrepeat containing protein pRBAP46/48 (10, 11). These subunitsrecruit NURF to chromatin through interactions with sequence-specific transcription factors ormodifiedhistones.Once recruited,NURF slides nucleosomes in cis to alter nucleosome positioningwithout histone eviction or exchange. These sliding reactions alterDNAaccessibility to regulate transcription factor binding, and as aresult gene expression (9).

Our previous studies demonstrate that BPTF preferentiallyregulates chromatin structure and the expression of the MHCgenes (5–7). These genes predominantly function to process andpresent antigens to immune cells. MHC genes with these func-tions include Psmb9 and Psmb8, which encode subunits of theimmunoproteasome, and Tap1 and Tap2, which compose thetransporter associated with antigen-processing (TAP) complex(12). The immunoproteasome is a specialized proteasome thatcreates peptides with higher affinity to MHC I molecules and istherefore more antigenic to CD8 T cells (12–14). Because geneslocated in the MHC are important for CD8 T-cell activity, they arefrequently repressed by tumor cells to avoid antitumor immunity(15). In an attempt to improve the immunogenicity of tumorcells, we depleted BPTF in the well-established B16F10 and 4T1tumormodels andmonitored for changes inMHCexpression andantitumor immunity.

1Department of Human and Molecular Genetics, Virginia Institute ofMolecular Medicine, Massey Cancer Center, Virginia CommonwealthUniversity, Richmond, Virginia. 2Department of Pathology, VirginiaCommonwealth University, Richmond, Virginia.

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

Corresponding Author: Joseph W. Landry, Virginia Commonwealth University,Goodwin Research Labs, 401 College Street, GRL-167, Richmond, VA 23298.Phone: 804-628-1944; Fax: 804-827-0810; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-3125

�2016 American Association for Cancer Research.

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Materials and MethodsMice

BALB/c, C57BL/6, NOD/SCID, Ifrg2r�/� (NSG), B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J (Jackson Laboratories), and OT1 (Giftfrom Dr. Shawn Wang, Virginia Commonwealth University,Richmond, VA) female mice 6–8 weeks of age were housed underaseptic conditions. These studies have been approved by theInstitutional Animal Care and Use Committee at Virginia Com-monwealth University.

Cell culture4T1 (Fred Miller, Wayne State University in 2010), B16F10

(ATCC in 2011), and HEK 293T (ATCC in 2010) were culturedin complete media [CM; DMEM (Life Sciences), 10% FBS(Hyclone), 1% NEAA, 2 mmol/L glutamine, and 1% penicil-lin/streptomycin (Life Technologies)]. T cells were cultured inCM with 10 mmol/L HEPES, 5 � 10�5 M ß-mercaptoethanol(Sigma) and 50 U/mL IL2 (R&D Systems). Hybridoma lineswere cultured in RPMI1640 (Life Sciences), 10% FBS, 1%NEAA, 2 mmol/L glutamine, and 1% penicillin/streptomycin.Population doubling times were calculated by standard meth-ods. Cell lines were authenticated by ATCC prior to shipmentby short tandem repeat (STR) profiling.

Short-hairpin RNAs (shRNA) were introduced into 4T1 andB16F10 cells using Moloney murine leukemia virus (MMLV)using the pSIREN-RetroQ (Clonetech) system. Transducedcells were selected with 0.5 mg/mL puromycin (Life Technolo-gies) after 48 hours. B16F10 cells were transfected with CMV-OVA along with a zeocin-selectable marker and selected with400 mg/mL zeocin (Invitrogen). Individual colonies werecloned and assayed for OVA expression, BPTF was thenknocked down (KD) with a shRNA in OVA-expressing lines.pSIREN plasmids Ctrl-sh1, Ctrl-sh2, Bptf-sh1 (4T1), Bptf-sh2(4T1), Bptf-sh1 (B16F10), and Bptf-sh2 (B16F10) are availableat Addgene as stock numbers 73665, 73666, 73669, 73668,73667, and 73669, respectively.

Tumor studiesA total of 1 � 104 4T1 cells were injected into the fourth

mammary fat pad of BALB/c or NSG mice and tumors wereanalyzed at 21 days. A total of 5� 104 B16F10 cells were injectedsubcutaneously into the flank of C57BL/6 mice and tumors wereanalyzed at 18 days. For gemcitabine-treatedmice, 1� 105 4T1 or5� 105 B16F10 cells were injected into BALB/c, NSG, or C57BL/6mice and 1.2 mg gemcitabine (Hospira) was injected intraper-itoneously (i.p.) on day 5 and every 7 days until tumors wereanalyzed on day 37 (4T1) or day 18 (B16F10).

mAb depletionsGK1.5 (anti-CD8), 2.43 (anti-CD4; gift from Dr. Mosoud

Manjili, Virginia Commonwealth University, Richmond, VA, in2012) and SH-34 (anti-asialo GM1; ATCC in 2013) mAbs werepurified from ascites fluid by ammonium sulfate fractionation(16). mAb (225 mg/mouse) was injected intraperitoneally intoBALB/c or C57BL/6 mice on day �2 and day �1 and mice wereinoculatedwith 1� 105 4T1or 5�104 B16F10 tumor cells onday0. mAb was injected once every 5 days following tumor inocu-lation and 4T1-bearing mice were treated with gemcitabine asdescribed previously. Hybridomas were authenticated by con-firming antigen-specific reactivity of produced antibodies by flow

cytometry using commercial anti-CD8, anti-CD4, anti-asialoGM1 antibody standards. Depletion of CD8, CD4 T cells, andNK cells from mice were confirmed by flow cytometry.

Western blot analysisProtein from cell cultures or homogenized tumors was

extracted by TRI Reagent (Sigma). Primary antibodies: anti-BPTF (Millipore), anti-OVALBUMIN, anti-PMEL17 (Santa CruzBiotechnology), anti-cyclophilin B (Abcam), anti-PSMB9,anti-TAP2 (Thermo Scientific), anti-PSMB8, and anti-TAP1(Cell Signaling Technology). Secondary antibody: horseradishperoxidase-conjugated anti-rabbit or anti-mouse secondaryantibody (Cell Signaling Technology).

Metastasis assayColony formation assay from the lungs of 4T1 tumor–bearing

mice were performed as described previously (17).

qRT-PCRTotal RNA was extracted from cultured cells with TRI Reagent

and 5 mg RNA was reverse transcribed using Superscript II First-Strand Kit (Invitrogen). qPCR was performed using SYBR GreenPCR master mix (Bio-Rad) with a 7900 HT Fast Real-Time qPCRSystem (Applied Biosystems). The DDCt method was performedusing normalization to Gapdh. Primer pairs are found in Sup-plementary Dataset S1.

Flow cytometryCultured 4T1 and B16F10 cells were stained with H2K, H2D,

Annexin V, OVA, and 7AAD viability dye. Mean fluorescenceintensity (MFI) was calculated using Cyflogic. To stain infiltratinglymphocytes, tumors were first digested with DNaseI and colla-genase and lymphocytes were purified using Percoll gradients asdescribed previously (18). Purified lymphocytes were subse-quently stained with CD8, CD69, TCRb, CD44, BTLA antibodies,and 7AAD viability dye. All antibodies were purchased from BDPharmingen.

Chromatin immunoprecipitationChIP was performed as described previously (7). Briefly, cells

were fixed in 1% paraformaldehyde for 15 minutes and chroma-tin was sheared to an average size of 300 bp by sonication. Lysateswere incubatedwith0.5mg anti-BPTF (Millipore) or control rabbitIgG bound to Protein G Dyna beads (Life Technologies) over-night, subsequently washed with low- and high-salt buffers andelutedwith 0.1mol/LNaHCO3 in 1%SDS. Primer pairs are foundin Supplementary Dataset S1.

CTL cytotoxicity assayCD8aþ cells were purified by negative selection using MACS

Separation Columns. The purified CTLs were restimulated withmitomycin C–treated 4T1 or B16F10 tumor cells. CTLs wereharvested, purified with by percoll gradient, and cocultured withmitomycin C–treated tumor cells in 96-well plates. For hyper-activation, purified splenic CD8aþ T cells were treated with0.8 mmol/L PMA, 0.35 mmol/L ionomycin, and 500 U/mL IL2on mitomycin C–treated tumor cells. For total splenocyte andOVA analysis, splenocytes from tumor-bearingmice or na€�veOT1mice were restimulated in vitro as described. For ONX-0914treatment, ONX-0914 was added to targets for 24 hours before

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Cancer Res; 76(21) November 1, 2016 Cancer Research6184




incubation with splenocytes at a 50:1 effector:target (E:T) ratio.For all assays, T cells/splenocytes were incubated on targets for 48hours and cell death was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega).

ELISPOT assayA total of 2� 106 B16F10 tumor-infiltrating lymphocytes were

cocultured with 1 � 106 splenocytes from na€�ve mice and stim-ulated with 1 mg/mL Gp10025–33 (KVPRNQDWL), TRP2180-188(SVYDFFVWL), or p15E604-611 (KSPWFTTL) and 50 U/mL IL2 for96 hours at 37�C in wells precoated with IFNg capture antibody.ELISPOT was performed as described previously (19).

Limited dilutionT-cell limited dilution was performed as described previously

(20). Cloneswere assayed for activity against control andBPTFKDtargets with the CytoTox 96 Non-Radioactive Cytotoxicity Assay(Promega).

Formaldehyde-assisted isolation of regulatory elementsFormaldehyde-assisted isolation of regulatory elements

(FAIRE) was performed essentially as described previously(21). FAIRE results were normalized to a control locus, whichdoes not have BPTF-dependent FAIRE when normalized to equalDNA mass. Primer pairs are found in Supplementary Dataset S1.

MicroarrayRNA extraction, microarray analysis, and statistical analysis

were performed as described previously (22, 23). Briefly, necrotictissue was selected out by macrodissection and total RNAwas extracted from frozen tumor tissues with TRI reagent Mag-MAX-96 Total RNA Isolation Kit. Biotinylated cRNA was gener-atedwith theGeneChip 3'IVT Express Kit and10mgwas applied to

the GeneChip Mouse Genome 430A 2.0 Array. Arrays werescanned using an Affymetrix GeneChip Scanner 3000 and datawere processed as described in GEO accession GSE71864.

ResultsBPTF depletion increases T-cell antitumor immunity to 4T1tumors

Consistent with previous work showing that subunits of theNURF complex (Fig. 1A) are frequently overexpressed in cancercells (8), we found each of the NURF subunits is expressed in avariety of mouse and human breast cancer cell lines (Supplemen-tary Fig. S1A). To discover functions of NURF in cancer cellbiology, we repressed BPTF expression in mouse 4T1 cells byretrovirus-introduced shRNA KD (Fig. 1B). In vitro, BPTF KD cellsshow no significant differences in doubling time, cellular mor-phology, or levels of apoptosis (Supplementary Fig. S1B–S1D).

To uncover functions for BPTF in tumor biology, we introducedBPTF KD 4T1 cells into the fourth mammary fat pad of syngeneicBALB/c mice. As part of our studies, we inoculated the cells intoboth untreated and gemcitabine-treated mice. Gemcitabine is aroutinely used chemotherapeutic that inhibits DNA replicationand induces apoptosis in tumors (16). We observed significantlyreduced weights for BPTF KD tumors only in gemcitabine-treatedmice (Fig. 1C). We chose weight and not volume to monitortumor growth because BPTF KD tumors are morphologically flatcompared with controls, confounding a volume-based measure-ment (Supplementary Fig. S2A and S2B).

In addition to inducing cancer cell apoptosis, gemcitabine alsoinhibits the proliferation of several cells of the immune system,most prominently myeloid-derived suppressor cells (MDSC;refs. 16, 24–27). MDSCs are dramatically amplified in micebearing 4T1 tumors, which suppresses the antitumor immuneresponse (25). To determine whether the effect of gemcitabine on

Figure 1.

Depletion of BPTF reduces 4T1 tumor weights in mice with CD8þ and CD4þ T cells. A, cartoon of NURF (BPTF, 2L, 48) bound to chromatin. Gray circles, histonemodifications; TF, transcription factor.B,BPTFWestern blot analysis fromcontrol (Ctrl-sh1, Ctrl-sh2) and BPTFKD (Bptf-sh1, Bptf-sh2) 4T1 total cell extracts. CyclophilinB, loading control. C and D, weights of primary control and BPTF KD 4T1 tumors after growth in BALB/c mice (C; n � 12; � , t test, P < 9.6 � 10�7) or NSG mice(D; n ¼ 9). E, weights of 4T1 tumors after growth in undepleted, CD8þ, CD4þ, or asialo-GM1þ mAb–depleted BALB/c mice (n � 5; � , t test, P < 0.02).

BPTF Regulates Antigen Processing

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BPTF KD tumors is a result of enhanced antitumor immunity,we repeated our tumor studies in immunocompromisedNOD/SCID, Ifrg2r�/� (NSG) mice (28). In NSG mice, weobserved similar growth of control and BPTF KD tumors withgemcitabine treatment (Fig. 1D), demonstrating that the reducedtumor growth in BALB/cmice is not due to gemcitabine alone, butrather in combination with antitumor immunity.

To determine whether BPTF KD cells are eliminated fromtumors grown in immunocompetent mice, we measured BPTFlevels in the primary tumors. We show that BPTF expressionincreases in tumors from BALB/c (Supplementary Fig. S2C) butnot NSG mice (Supplementary Fig. S2D), suggesting that afunctional immune system either directly or indirectly selects forcells that re-express BPTF.

To understand the immune response to gemcitabine-treatedBPTF KD 4T1 tumors, we selectively depleted CD8þ T cells,CD4þ T cells, or NK cells by antibody-dependent cellularcytotoxicity (ADCC). We observed a rescue of BPTF KD tumorgrowth when CD8þ or CD4þ cells were depleted (Fig. 1E),demonstrating that the antitumor response to BPTF KD 4T1tumors requires T cells.

MDSC amplification and tumor metastasis are BPTFindependent

4T1 tumors dramatically amplify MDSCs, resulting in spleno-megaly with spleen size proportional to the degree of MDSCamplification (25). As previously reported, we observed a signif-icant increase in spleen weight with introduction of 4T1 tumorsinto BALB/c mice (Supplementary Fig. S2E). The observedincrease in spleen weight was proportional to tumor weight andwas not affected by BPTF KD, demonstrating that splenomegaly isBPTF independent (Supplementary Fig. S2F).

The 4T1 model is also widely used to study breast cancer cellmetastasis (17). To determine whether BPTF regulates metastasis,we analyzed metastases to the lung of both untreated and gemci-tabine-treated BALB/c mice using a colony formation assay (17).We show that tumor size, but not BPTF KD, was significantlycorrelatedwith the number of colonies, suggesting thatmetastasisof 4T1 is BPTF independent (Supplementary Fig. S2G and S2H).

BPTF depletion increases T-cell antitumor immunity to B16F10tumors

To further verify our findings, we used the B16F10 melanomamodel, which is syngeneic to C57BL/6 (29). First, we confirmedthat each NURF subunit is expressed in B16F10 cells (Supple-mentary Fig. S3A). In addition, BPTF KD (Fig. 2A) had nosignificant effect on doubling time, cellular morphology, orapoptosis of B16F10 cells (Supplementary Fig. S3B–S3D).

Consistent with 4T1, B16F10 KD tumors had a flattenedmorphology, preventing caliper measurements from accuratelycomparing tumor growth (Supplementary Fig. S3E and S3F). Aswith 4T1, we observed significant reductions in BPTF KD B16F10tumor weights (Fig. 2B). However, unlike in the 4T1 model,gemcitabine treatment did not selectively reduce BPTF KDB16F10 tumor growth (Supplementary Fig. S3G).

To determine whether CD4 and CD8 T cells are required forreduced B16F10 BPTF KD tumor growth, we repeated our ADCCexperiments. Consistent with an enhanced T-cell response, weobserved a rescueof BPTFKD tumor growthwithCD4þ andCD8þ

cell depletion, but notNK-cell depletion (Fig. 2C). These results incombination support a model of an enhanced CD8 T-cell cyto-

toxic response to BPTF KD tumors, which requires CD4 Th cellactivity.

CD8 T cells are more abundant and activated in BPTF KDtumors

We then measured the infiltration and activation status ofintratumoral CD8 T cells by flow cytometry. As expected, weobserved a greater number of CD8 T cells (CD8þ, TCRbþ) in theBPTF KD tumor microenvironment (Fig. 3A and B and Supple-mentary Fig. S4). We next measured the abundance of activationmarkers CD69 and CD44 and the anergy marker BTLA on intra-tumoral CD8 T cells. We observed a greater percentage ofCD69high CD8þ cells in both 4T1 and B16F10 BPTF KD tumors(Fig. 3C and D). In contrast, CD44 and BTLA expression differedsignificantly on CD8 T cells between control and BPTF KDB16F10, but not 4T1, tumors (Fig. 3E–H). This could be theresult of differences in the tumormicroenvironment between 4T1and B16F10 (see Discussion). Together, these results indicate thatthe BPTF KD tumor microenvironment has a greater number ofactive CD8 T cells.

CD8 T cells have enhanced cytotoxic activity toward BPTF KDcells in vitro

To investigate whether BPTF KD 4T1 and B16F10 tumor cellsare more efficiently targeted by CD8 T cells, we performed in vitrocytotoxicity assays. Coculture of purified splenic CD8 T cellsisolated from BPTF KD tumor–bearingmice with BPTF KD targets

Figure 2.

BPTF depletion reduces B16F10 tumor weights in mice with CD8þ and CD4þ Tcells. A, BPTF Western blot analysis of control (Ctrl-sh1, Ctrl-sh2) and BPTF KD(Bptf-sh1, Bptf-sh2) B16F10 total cell extracts. Cyclophilin B, loading control.B, weights of primary control and BPTF KD B16F10 tumors after growth inC57BL/6 mice (n� 9; � , t test, P < 1.4� 10�7). C,weights of B16F10 tumors aftergrowth in undepleted, CD8þ, CD4þ, or asialo-GM1þ mAb–depleted C57BL/6mice (n � 5; � , t test, P < 0.02).

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results in enhanced cytolytic activity compared with similarexperiments using controls (Fig. 4A).

We next determined whether BPTF KD cells are more suscep-tible to T-cell–induced cell death. Toward this end, we used PMAþ ionomycin–activated na€�ve CD8 T cells and observed similarcytolytic activity between control and BPTF KD targets (Fig. 4B).These results demonstrate that enhanced CD8 T-cell killingof BPTF KD target cells was not due to increased sensitivity toCD8 T-cell–mediated cell death.

To investigate whether BPTF KD cells are more antigenic thancontrol cells, we used the OT1 and pmel CD8 T-cell TCR (T-cellreceptor) transgenic models (30, 31). The pmel TCR recognizespeptides from the endogenously expressed Pmel, whereas the OT1TCR recognizespeptides fromchickenovalbumin (OVA)presentedby H2-Kb, which is expressed on B16F10, but not 4T1 (30, 31).

Coculture experiments show that neither pmel nor OT1 CD8T cells had enhanced reactivity to BPTF KD B16F10 cells (Sup-plementary Fig. S5A and S5B) even though they expressed equiv-alent OVA and increased PMEL protein (Supplementary Fig. S5Cand S5D). Consistent with BPTF-independent OVA antigen pre-sentation, an antibody that recognizes the OVA peptide (SIIN-FEKL) in context withH2-Kb (32), equivalently stains control andBPTF KD B16F10 cells (Supplementary Fig. S5E). To furthercharacterize the CD8 T-cell response to known antigens, wequantified gp100, TRP2, and p15E-reactive intratumoral CD8T cells by ELISPOT. From this, we observed similar numbers ofpeptide-reactiveCD8T cells between control andBPTFKD tumors(Supplementary Fig. S5F). Together, these results suggest thatenhanced CD8 T-cell activity to BPTF KD tumors occurs to novelantigens.

To investigatewhether BPTFKDcells present novel antigens,wecloned CD8 T cells from both BPTF KD and control tumor–bearing mice. Coculture assays showed enhanced activity of CD8T-cell clones from BPTF KD tumor–bearing mice to BPTF KDtarget cells compared with similar experiments using controls(Fig. 4C). Furthermore, this enhanced activity usually occurs only

when cocultured with BPTF KD, but not control, target cells (fiveof nine assayed clones; Fig. 4D). In contrast, we do not observeenhanced cytolytic activitywhenCD8T cells isolated fromcontroltumor–bearingmice are coculturedwith BPTFKD target cells (0 of4 assayed clones; Fig. 4D). These results support the hypothesisthat BPTF KD tumors express novel antigens.

BPTF directly regulates antigen processingTo identify BPTF-dependent genes that could alter tumor cell

antigenicity, we performed genome-wide expression arrays on4T1 tumors harvested fromNSGmice (Fig. 5A). We observed 115upregulated genes and 199 downregulated genes (>1.5 foldchange in expression, FDR < 0.05; Fig. 5A and SupplementaryDataset S1 and Supplementary Table S1). Of genes identified, wefocused on Psmb9 because it regulates antigenicity as a subunit ofthe immunoproteasome (33). We confirmed Psmb9 upregulationin vitro, and observed elevated expression of the neighboringantigen-processing genes Psmb8, Tap1, and Tap2 in both tumormodels (Fig. 5B andC). The upregulation of these genes correlateswith equivalent to slight increases in cell surface expression ofH2KorH2D inBPTFKDcells asmeasured byMFI, whichwere notthe result of increased gene expression (Fig. 5D–F). In addition,the expression of the IFN-inducible genes Oas1a and Oas2 werenot upregulated with BPTF KD, indicating that upregulation ofPsmb8, Psmb9, Tap1, and Tap2 are not a result of a general IFNresponse (Supplementary Fig. S6A).

To determine whether BPTF directly regulates the expression ofPsmb8, Psmb9, Tap1, and Tap2, we measured BPTF occupancy bychromatin immunoprecipitation (ChIP; ref. 34). Sites chosen forChIP were guided by previously identified DNaseI hypersensitiv-ity hotspots, and therefore possible regulatory elements, fromgenome-wide studies done in the 3,134 mouse mammary epi-thelial cell line (Fig. 6A; ref. 35). The most consistent enrichmentof BPTF between 4T1 and B16F10 was detected at the promoters,consistent with BPTF directly repressing these genes (Fig. 6B andSupplementary Fig. S6B and S6C). To determine whether BPTF

Figure 3.

Enhanced presence and activity of CD8 T cells in BPTF KD tumors.A, C, E, and G, representative dot plots of live 4T1 tumor–infiltrating lymphocytes stained for CD8and TCRb, CD69, CD44, or BTLA, respectively. B, percentages of live intratumor CD8þ, TCRbþ lymphocytes as a percent of all live lymphocytes from 4T1 andB16F10 tumors (n� 7; � , t test, P <0.05).D, F, andH, percentages of intratumor CD8þ lymphocytes that are CD69high, CD44þ, or BTLAþ from 4T1 and B16F10 tumors,respectively (n ¼ 6; � , t test, P < 0.05).






chromatin-remodeling activity could be relevant to changes ingene expression,we focusedon thewell-characterizedPsmb9-Tap1divergent promoter (36), using formaldehyde-assisted isolationof regulatory elements (FAIRE; ref. 21).With this technique, openchromatin is isolated fromfixed cells usingphenol extractions andquantified by qPCR relative to a BPTF-independent reference site.FAIRE shows that BPTF maintains chromatin structure of thePsmb9-Tap1 promoter in both cell lines (Fig. 6C).

To verify that increased expression of the immunoproteasomesubunits is responsible for the enhanced antigenicity of BPTF KDcells, we utilized the PSMB8-selective inhibitor ONX-0914 (37).Cytotoxicity assays show that enhanced CD8 T-cell cytotoxicityto BPTF KD targets is ablated after treatment with ONX-0914(Fig. 6D). These results allow us to propose a model where BPTF

depletion upregulates the antigen-processing genes Psmb8, Psmb9,Tap1, and Tap2, which results in enhanced antigenicity andimproved T-cell antitumor immunity. As a corollary, we proposethe BPTF normally suppresses antitumor immunity by repressingantigen processing in cancer cells.

DiscussionUsing KD of BPTF as ameans to deplete the NURF complex, we

show that it suppresses T-cell antitumor immunity in two mousetumor models with different genetic backgrounds. IntratumorCD8 T cells are more active in BPTF KD tumors. Differencesbetween the tumor models include CD44 and BTLA expression.This is partially the result ofMDSCactivity in 4T1 tumors,which is

Figure 4.

BPTF depletion sensitizes tumor cells toCD8 T-cell cytotoxicity in vitro. A–D,percent target cell cytotoxicitydetermined by LDH release. A, purifiedsplenic CD8 T cells from control or BPTFKD tumor–bearing mice were coculturedon control or BPTF KD targets,respectively, at the indicated E:T ratios(n ¼ 3; � , t test P < 0.05). B, coculture ofna€�ve purified CD8 T cells treated withPMA þ ionomycin with 4T1 or B16F10targets at a 10:1 E:T ratio. C, CD8 T-cellclones isolated from spleens of 4T1control or BPTF KD tumor–bearing micewere cocultured with 4T1 control or BPTFKD targets, respectively, at a 10:1 E:T ratio.Each dot represents one clone and is anaverage of three biological replicates(� , t test, P < 5.0 � 10�3). D, sixrepresentative CD8 T-cell clones fromC were cocultured with either control orBPTF KD 4T1 targets at a 10:1 E:T ratio.Results are representative of threebiological replicates for each clone(� , t test, P < 0.04).

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known to suppress CD44, but not CD69, abundance on CD8 Tcells (38). Differences in BTLA could result because the 4T1 tumormicroenvironment has elevated costimulatory signals comparedwithB16F10, preventing T-cell anergy (39). It is unlikely that BPTFregulates costimulatory molecules on the tumor because not allCD8 T-cell clones are preferentially reactive to BPTF KD targets.Our in vitro assays reveal that BPTF protects tumor cells fromdirectCD8 T-cell cytotoxic activity. Enhanced antitumor activity to BPTFKD cells is observed only when CD8 T cells are primed andactivated with BPTF KD tumor cells, suggesting that BPTF sup-presses antigen presentation. It is not likely that BPTF is a generalregulator of antigen presentation because we observe approxi-mately equivalent levels of cell surface H2K and H2D with BPTFKD. The degree to which an antigen stimulates T-cell activitydepends on both the number of an antigen:MHC complex pre-sented and the quality of the antigen (40, 41). We utilized OT1and pmel transgenic CD8 T cells to determine whether BPTFsuppresses the presentation of known antigens (30, 31). Despiteequal or enhanced expression ofOVAor PMEL antigens, coculture

experiments show that BPTF does not lend protection againstOVA- and pmel-reactive CD8 T cells. These results indicate thatBPTF represses the presentation of select antigens to CD8 T cells.Consistent with this, assay of CD8 T-cell clones isolated from 4T1BPTF KD tumor–bearing mice identified many clones withenhanced reactivity specifically toward BPTF KD targets. Theidentity of these antigens is currently unknown, but they likelyresult from BPTF regulating presentation of tumor-specific anti-gens (TSA) or tumor-associated antigens (TAA; ref. 42). Ourmicroarray analysis of 4T1 tumors did not reveal any known TSA,but these results would not detect differences in expression ofalloantigens. The 4T1 tumor line has a BALB/cfC3H hybridgenotype, and therefore presents alleles from the C3H back-ground, which would be recognized as alloantigens in BALB/cmice (17).

In addition to the presentation of TAA and TSA, novel antigenscan be created by changes in antigen processing. From ourexperiments, we observe that BPTF represses the expression ofPsmb8, Psmb9, Tap1, and Tap2. Upregulation of the

Figure 5.

BPTF regulates immunoproteasome and TAP subunit expression. A, microarray heatmap of genes significantly deregulated in BPTF KD 4T1 tumors from NSGmice (n ¼ 3). Scale represents � 3-fold expression change. Genes related to the immune response are highlighted. B, qRT-PCR analysis of Psmb8, Psmb9, Tap1,and Tap2 expression from control or BPTF KD 4T1 and B16F10 cells (n ¼ 3 biological replicates; �, t test, P < 0.05). C, PSMB8, PSMB9, TAP1, and TAP2Western blot analysis of 4T1 (top) and B16F10 (bottom) total cell extracts. D, representative flow cytometry histograms of 4T1 and B16F10 cells stainedfor theMHCclass ImoleculesH2KandH2D.E, fold change inMFI ofH2KandH2D (n�3biological replicates; � , t test,P<0.05).F,qRT-PCRanalysis ofMHCclass I geneexpression from 4T1 and B16F10 cells (n ¼ 3 biological replicates).






Figure 6.

BPTF occupies and regulates chromatin structure at Psmbs and TAPs. A, schematic showing the position of PCR amplicons used for ChIP analysis and DNaseIhotspots from themousemammary epithelial line 3134 (35).B,BPTF ChIP at Psmb9, Psmb8, Tap1, and Tap2 promoters (n¼ 3 biological replicates; � , t test, P <0.05).C, FAIRE at the divergent Psmb9-Tap1 promoter. Values are normalized to a BPTF-independent control site (n ¼ 3 biological replicates; � , t test, P < 0.05).D, percent target cell cytotoxicity determined by LDH release for 4T1 splenocytes from control or BPTF KD tumor–bearing mice stimulated at a 50:1 E:T ratio oncontrol or BPTF KD targets, respectively, after treatment with 50 to 200 nmol/L ONX-0914 (n ¼ 3 biological replicates; � , t test, P < 0.04).

Mayes et al.





immunoproteasome subunits Psmb8 and Psmb9with BPTF deple-tion would result in increased immunoproteasome activity, gen-eratingmore antigenic peptides (14, 33, 43). Peptides with greaterantigenicity can more favorably bind to MHC molecules orpromote higher affinity interactions with the TCR (40). In addi-tion, upregulation of the TAPs alters the repertoire of peptidespresented by changing the types of peptides transported into theER for loading into MHC molecules (13). BPTF could directlyregulate these genes because it is localized to the Psmb and Tappromoters in both cell lines. BPTF also occupies distal elements,most significantly in 4T1, which could also influence gene expres-sion. BPTF, presumably throughNURF, could remodel chromatinstructure at either of these locations to repress gene expression.This is supported by anobserved change in chromatin structure byFAIRE at the Psmb9-Tap1 promoter with BPTF KD. Psmb8, Psmb9,Tap1, and Tap2 promoters are regulated by IFNg through theSTAT1 and IRF-1 transcription factors (44, 45), and are repressedin tumor cells by DNA methylation and histone deacetylation(46–48). Our finding that BPTF represses their expression, pre-sumably through the NURF complex, presents a novel epigeneticmechanism to suppress tumor cell antigenicity.

The NURF subunits are rarely deleted and frequently amplifiedor overexpressed in cancer cells, suggesting that a majority oftumor cells will have a NURF complex to inhibit (8, 49). BPTF isnot necessary for the viability of any primary cell type examined(5–7), suggesting that NURF inhibition may be tolerated inadults. Therefore, it is plausible that inhibiting NURF could bea viable strategy to improve tumor cell antigenicity. Furthermore,NURF is an enzyme with several substrate binding sites (DNA,histones, and ATP) and critical interaction surfaces with chroma-tin (transcription factors, DNA, and modified histones), each ofwhich could be targeted by small molecules (9).

As with most therapies, a NURF inhibitor would be moreeffective when used in combination with other chemo or immu-notherapies.BPTFdepletion improves antigenicity, but these effectsare only relevant for antitumor immunity whenMDSC abundanceis low.Hence, the need to use gemcitabine to depleteMDSCs in the

4T1model. In contrast, the B16F10model has low levels ofMDSC,not requiring the use of gemcitabine (50). The contrast betweenthese two tumormodelsprovidesproofofprinciple for theutilityofa NURF inhibitor in combinatorial therapeutic regimens.

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

Authors' ContributionsConception and design: K. Mayes, S.G. Alkhatib, J.W. LandryDevelopment of methodology: K. Mayes, S.G. Alkhatib, C.I. Dumur,J.W. LandryAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Mayes, S.G. Alkhatib, K. Peterson, A. Alhazmi,V. Chan, T. Blevins, M. Roberts, C.I. Dumur, J.W. LandryAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Mayes, S.G. Alkhatib, C.I. Dumur, X.-Y. Wang,J.W. LandryWriting, review, and/or revision of the manuscript: K. Mayes, S.G. Alkhatib,X.-Y. Wang, J.W. LandryAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):K.Mayes, S.G. Alkhatib, K. Peterson, A. Alhazmi,C. Song, V. Chan, T. Blevins, C.I. Dumur, J.W. LandryStudy supervision: C.I. Dumur, J.W. Landry

AcknowledgmentsThe authors would like to thank Joyce Lloyd, Masoud Manjili, Tomas

Kordula, and Chunqing Guo for critically reading the manuscript.

Grant SupportThese experiments were funded by start-up funds from the Department

of Human and Molecular Genetics (J.W. Landry), the Massey Cancer Center(J.W. Landry), and an early-stage investigator grant from the V Foundation forCancer Research (J.W. Landry).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 17, 2015; revised August 12, 2016; accepted August 24,2016; published OnlineFirst September 20, 2016.

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2016;76:6183-6192. Published OnlineFirst September 20, 2016.Cancer Res Kimberly Mayes, Suehyb G. Alkhatib, Kristen Peterson, et al.

Mediated Antitumor Immunity−BPTF Depletion Enhances T-cell

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