Immunosuppressive Microenvironment and Sensitizes FXR PD ... · Introduction Non–small cell lung cancer (NSCLC), which constitutes about ... 1Department of Respiratory Medicine,

Research Article

Farnesoid X Receptor Constructs anImmunosuppressive Microenvironment andSensitizes FXRhighPD-L1low NSCLC to Anti–PD-1ImmunotherapyWenjie You1,2, Lijun Li3,4, Deqiao Sun3,4,5, Xueqing Liu1, Zongjun Xia3,4, Shan Xue1,Bi Chen6, Hui Qin1, Jing Ai3,4, and Handong Jiang1

Abstract

The farnesoid X receptor (FXR) regulates inflammation andimmune responses in a subset of immune-mediated diseases.We previously reported that FXR expression promotes tumorcell proliferation in non–small cell lung cancer (NSCLC). Herewe study the relevance of FXR to the immunemicroenvironmentof NSCLC. We found an inverse correlation between FXR andPD-L1expression ina cohortof408NSCLCspecimens; fromthis,we identified a subgroup of FXRhighPD-L1low patients. Weshowed that FXR downregulates PD-L1 via transrepression andother mechanisms in NSCLC. Cocultured with FXRhighPD-L1low

NSCLC cell lines, effector function and proliferation of CD8þ

T cell in vitro are repressed. We also detected downregulationof PD-L1 in FXR-overexpressing Lewis lung carcinoma (LLC)mouse syngeneic models, indicating an FXRhighPD-L1low sub-type in which FXR suppresses tumor-infiltrating immunecells. Anti–PD-1 therapy was effective against FXRhighPD-L1low

mouse LLC tumors. Altogether, our findings demonstrate animmunosuppressive role for FXR in the FXRhighPD-L1lowNSCLCsubtype and provide translational insights into therapeuticresponse in PD-L1low NSCLC patients treated with anti–PD-1.We recommend FXRhighPD-L1low as a biomarker to predictresponsiveness to anti–PD-1 immunotherapy.

IntroductionNon–small cell lung cancer (NSCLC), which constitutes about

85% of all lung cancers, remains a leading cause of cancermortality worldwide (1). The identification of tumor oncogenicgene alterations has transformed the management of NSCLC,leading to responses in selected patients treated with matchedtyrosine-kinase inhibitors (2). Moreover, the tumor microenvi-ronment (TME) emerged as a target for anticancer therapydevelopment, because the interaction between tumor cells andstromal tumor–promoting immune cells also plays a role in

NSCLC progression (3). Progress in immunotherapy, targetingthe tumor-promoting immunosuppressive microenvironment,has been promising for treatment of cancers including NSCLC.However, many issues remain to be addressed in immunother-apy, especially in broadening the range of patients who canbenefit from immunotherapies.

Farnesoid X receptor (FXR) is a member of the nuclear receptorsuperfamily and is expressed in various tissues including liver,intestine, kidneys, and adrenal gland (4). As a ligand-activatedtranscription factor, FXR regulates expression of target genesinvolved in enterohepatic circulation and lipid homeostasis (5).However, emerging evidence has indicated the relevance of FXR intumorigenesis. FXRdeficiency inmice leads to increased colon cellproliferation and spontaneous liver tumors (6, 7). On the otherhand, FXR has a causative role in esophageal cancer, breast cancer,and pancreatic cancer (8–10). We previously reported that FXR isupregulated in NSCLC, compared with pericarcinous lung tis-sues (11). Our data also showed that FXR contributes to NSCLCcell proliferation via transactivating CCND1, suggesting anoncogenic role for FXR in NSCLC progression.

FXR has been implicated as a regulator of inflammation andimmune responses in a subset of immune-mediated disorders.For example, activation of FXR reduced immune cell infiltrationand expression of inflammatory mediators (e.g., MCP-1, IL1b,IL6, and IFNg) in animal models of nonalcoholic steatohepatitisor colitis (12, 13). Zhang and colleagues showed that expressionof FXR repressed proinflammatory genes and improved lungpermeability in FXR�/� mouse acute lung injury models (14),implying that FXR might contribute to NSCLC progression bymodulating the immune microenvironment as well. In the sameset of NSCLC samples as used in our previous work (11), we

1Department of Respiratory Medicine, Ren Ji Hospital, School of Medicine,Shanghai Jiao Tong University, Shanghai, China. 2Department of RespiratoryMedicine, Shandong Provincial Hospital affiliated to Shandong University, Jinan,China. 3Division of Antitumor Pharmacology, State Key Laboratory of DrugResearch, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,Shanghai, China. 4University of Chinese Academy of Sciences, Beijing, China.5School of Life Science and Technology, ShanghaiTech University, Shanghai,China. 6Department of Respiratory Medicine, Affiliated Hospital of XuzhouMedical University, Xuzhou, China.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

W. You, L. Li, and D. Sun contributed equally to this article.

Corresponding Authors: Handong Jiang, Ren Ji Hospital, School of Medicine,Shanghai Jiao Tong University, 160 Pujian Road, Shanghai 200123, China.Phone: 86-21-68385507; E-mail: [email protected]; and Jing Ai, ShanghaiInstitute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,China. Phone: 86-21-50806600, ext 2413; E-mail: [email protected]

Cancer Immunol Res 2019;7:990–1000

doi: 10.1158/2326-6066.CIR-17-0672

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observed a negative correlation between FXR expression andexpression of the checkpoint PD-L1, which orchestratesimmunosuppression in cancer. We identified a subgroup ofFXRhighPD-L1low NSCLC patients (Supplementary Table S1). Inthis study, we sought to determine whether FXR affects the tumorimmune microenvironment of NSCLC, especially in the contextof FXRhighPD-L1low tumors. Our study provides insights into theimmunosuppressive role for FXR in the FXRhighPD-L1low NSCLCsubtype. We recommend FXRhighPD-L1low status as a biomarkerfor guiding anti–PD-1 immunotherapy.

Materials and MethodsCell lines

Human NSCLC cell lines NCI-H1299, HCC4006, A549, andNCI-H1975, mouse Lewis lung carcinoma (LLC) cell line, mousecolon carcinoma (MC38) cell line, and mouse melonoma (S91)cell line were obtained from the ATCC in 2003 (LLC), 2011(A549), 2013 (NCI-H1299 and HCC4006), and 2015 (MC38,NCI-H1975, and S91). HCC827 cells were obtained from theInstitute of Biochemistry and Cell Biology, Chinese Academy ofSciences in 2010. All cell lines used in this study were culturedaccording to the suppliers' instructions. Cells were checked to beMycoplasma-free and passaged no more than 25 to 30 times afterthawing. Cell lines were characterized by Genesky BiopharmTechnology using short tandem repeat markers (latest tested in2017).

Patients and tissue microarraysFrom 2008 to 2010, 156 NSCLC samples andmatched normal

lung tissues with complete clinical and follow-up informationwere consecutively collected from theDepartment of Pathology inRen Ji Hospital (Shanghai, China). Another 74 NSCLC sampleswithout clinicopathologic or survival information were also col-lected from Ren Ji Hospital. Tissue microarrays were constructedas in our previous study (11). Two cores with a 1.6-mm diameterwere obtained from the original paraffin block of each sample.Patients who had received neoadjuvant chemotherapy or radio-therapywere excluded. Tissuemicroarrays containing 178NSCLCsamples and matched normal lung tissues were obtainedfrom Outdo Biotech Co. Ltd (product ID: HLugA180Su03and HLugS150Su02) and ZuoChengBio Co. Ltd (product ID:LUC1505). A total of 408 NSCLC cases were enrolled. This studywas approved by the Ethics Committee of Ren Ji Hospital. Allexperiments were conducted according to the Declaration ofHelsinki principles, as well as the approved guidelines of Schoolof Medical Graduate Shanghai Jiao Tong University. Writteninformed consent was obtained from all subjects.

IHC analysisThe tissue microarrays and paraffin-embedded mouse tumors

were subjected to IHC staining as previously described (15). Forhuman and mouse FXR detection, anti-NR1H4 (Sigma-Aldrich)was applied at 1:100 and 1:200, respectively. For human andmouse PD-L1 detection, anti–PD-L1 (Zeta Life) was applied at1:50. Isotype controls were conducted simultaneously usingconcentration-matched nonspecific rabbit IgG. The IHC stainingresults were independently evaluated by two trained pathologists.Staining intensity of tumor cells was scored 0 through 3 asnegative (0), weak (1), moderate (2), and intense (3). Thepercentage of positive cells was binned as 0 through 4 as 0%

(0), 1% to 25% (1), 26% to 50% (2), 51% to 75%(3), and 76% to100% (4). The final IHC score was themultiplication of these twoscores (range, 0–12).HumanFXR expression in tissuemicroarrayswas defined as low (score 0–4) or high (score 6–12), and humanPD-L1 in tissue microarrays defined as low (score 0–2) or high(score 3–12), respectively (11, 16).

siRNA transfectionTransfection was carried out using Lipofectamine RNAiMAX

reagent (Invitrogen) according to the manufacturer's protocol.The target sequences of double-stranded nucleotides usedin siRNA transfection were 50-GGACCATGAAGACCAGATT-30

(FXRsiRNA-1) and 50-GACCTCGACAACAAAGTCA-30 (FXRsiRNA-2)for FXR knockdown, 50-TCGCCCTATCATTGGAGAT-30 (SHPsiRNA-1), 50-CCAGCTATGTGCACCTCAT-30 (SHPsiRNA-2) and 50-CCA-AGATGCTGTGACCTTT-30 (SHPsiRNA-3) for small heterodimerpartner (SHP) knockdown in NSCLC cells; 50-CCAAGAACGC-CGTGTACAA-30 (mouse FXRsiRNA-1) and 50-GCGTGATG-GACATGTACAT-30 (mouse FXRsiRNA-2) for FXR knockdown inmouse S91 cells. The target sequence of negative control (NC)was50-TTCTCCGAACGTGTCACGT-30 (RiboBio). To inhibit the activ-ity of phospho-EGFR, H1975 and HCC827 cells were incubatedwith afatinib (0.8 mmol/L) or gefitinib (0.02 mmol/L, SelleckChemicals) 24 hours after transfection, respectively.

Lentiviral vectors and cell infectionLentiviral vectors were generated with OBiO Biotechnology,

using pCMV-NR1H4-PGK-PuroR or pCMV-mNR1H4-PGK-PuroR plasmids, which carry the full-length human NR1H4(GenBank accession NM_005123.3) ormouseNR1H4 (GenBankaccession NM_009108.2) coding sequence, respectively. Cellswere infected with lentiviral vectors as in our previous study (11).

Coculture assayA549 and LLC cells overexpressing FXR were seeded into

96-well plates (8� 103/well) overnight. Human peripheral bloodmononuclear cells (PBMC;HemaCare) were used for CD8þ T-cellsorting via magnetic sorting techniques (Miltenyi Biotec). Mousesplenocytes from7-week-oldC57BL/6 femalemicewere also usedfor CD8þ T-cell sorting magnetically (Stemcell Technologies).Human PBMCs, mouse splenocytes, and corresponding CD8þ Tcells were activated by plate-bound anti-CD3 (OKT3; 2 mg/mL,BioLegend), soluble anti-CD28 (1 mg/mL, BioLegend) mAbs andIL2 (5ng/mL, PeproTech), and then added to the attachedA549orLLC in triplicate at an effector-to-target ratio of 25:1. After 3 days,PBMCs, splenocytes, and CD8þ T cells were harvested from thecoculture system, stained with CD8-APC-H7 (BD Biosciences),IFNg-APC and TNFa-PE (eBioscience), and analyzed by flowcytometry. The proliferation of cocultured CD8þ T cells wasexamined using the CellTrace Violet Cell Proliferation Kit (Invi-trogen Corporation).

Flow cytometryFor in vitro analysis, cells were harvested and incubated with

fluorochrome-linked antibodies for 30 minutes at 4�C. Matchednonspecific mouse/rat IgG served as an isotype control. Afterbeing washed two times, cells were analyzed using a Calibur flowcytometer (BD Biosciences) equipped with FlowJo version 10software. For in vivo studies, mouse tumors were minced anddigestedusing theMouse TumorDissociationKit (Miltenyi). Cellswere passed through a 70-mm cell strainer, and single-cell

FXR Constructs Immunosuppressive Microenvironment

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suspensions were then analyzed by Aria III flow cytometry(BD Biosciences) as described above, with Fixable ViabilityStain 510 (BD Biosciences) to discriminate viable and deadcells. The following fluorochrome-linked antibodies wereused in this study. Anti-human: PD-L1–PE (BioLegend).Anti-mouse: CD45-PerCP-Cy5.5, CD45-APC-Cy7, GranzymeB-PE-Cy7, TNFa-PE, IFNg-APC, PD1-PE, Lag3-APC, F4/80-BV421, CD206-APC, CD3e-FITC, CD4-PE-Cy7, CD25-APC,Foxp3-PE (eBioscience), Fixable Viability Stain 510, CD8-APC-H7,CD49b-BV421, CD69-BV711, CD11b-FITC, CD11c-BV650,CD86-PE, MHC II-BV650, PD-L1-BV786 (BD Biosciences), andCD11c-PE-Cy7, Gr-1-PE, MHC II-PerCP-Cy5.5 (BioLegend).Fixation/permeabilization buffers (eBioscience) were used forintracellular staining.

Western blotWestern blot analysis was performed as previously

described (11). Anti-human bile acid receptor (NR1H4; Abcam),anti-SHP (Santa Cruz Biotechnology), anti-phospho-EGF recep-tor (Tyr1068), anti-EGF receptor, anti-phospho-Akt (Ser473),anti-Akt, anti-phospho-p44/p42MAPK(Erk1/2; Thr202/Tyr204),anti-p44/p42 MAPK (Erk1/2), anti–b-actin (Cell Signaling Tech-nology), and anti-mouse bile acid receptor (NR1H4; Abcam)were used according to the manufacturer's recommendeddilutions.

Quantitative real-time PCR (RT-PCR) analysisQuantitative RT-PCR was carried out as previously describ-

ed (11). b-Actin mRNA was amplified as an internal control.Primers were designed as follows: human FXR forward, 50-GATT-GCTTTGCTGAAAGGGTC-30; reverse, 50-CAGAATGCCCAGAC-GGAAG-30; SHP forward, 50-AGGCCTCCAAGCCGCCTCCCA-CATTGGGC-30; reverse, 50-GCAGGCTGGTCGGAAACTTGAGG-GT-30; PD-L1 forward, 50-CAATGTGACCAGCACACTGAGAA-30;reverse, 50-GGCATAATAAGATGGCTCCCAGAA-30; b-actin forward,50-TTGCTGATCCACATCTGCT-30; reverse, 50-GACAGGATGCAGA-AGGAGAT-30; mouse FXR forward, 50-TGTGAGGGCTGCAAAGGT-TT-30; reverse, 50-ACATCCCCATCTCTCTGCAC-30; PD-L1 forward,50-GACCAGCTTTTGAAGGGAAATG-30; reverse, 50-CTGGTTGATT-TTGCGGTATGG-30;b-actin forward, 50-GGCATTGTTACCAACTGG-GACGAC-30; reverse, 50-CCAGAGGCATACAGGGACAGCACAG-30.

Chromatin immunoprecipitation (ChIP) assayThe ChIP assays were conducted using a SimpleChIP Plus

Enzymatic Chromatin IP Kit (Cell Signaling Technology) as inour previous study (11). ChIP primers for PD-L1 promoter weredesigned as follows: forward 50-CACCCATACACACACACACAC-30; reverse 50-ACAACAAGCCAACATCTGAAC-30.

Luciferase reporter assayLuciferase reporter plasmids carrying either wild-type or

FXR-responsive element (FXRE)-deleted PD-L1 promoter se-quence were constructed by OBiO Biotechnology. The luciferasereporter assays were performed using a Dual-Luciferase ReporterAssay System (Promega) as our previous study (11). The fireflyluciferase value was normalized to the corresponding Renillaluciferase value in order to compare the transfection efficiency.

Animal experimentsFemale C57BL/6 mice (4–6-week-old) were housed and

maintained under specific pathogen-free conditions. To generate

subcutaneous xenograft tumors, LLC-Mock or LLC-FXR cells(1� 106) were suspended in 100 mL of phosphate-buffered salineand inoculated subcutaneously (s.c.) into the right flanks ofC57BL/6 mice. Subsequently, the well-developed tumors werecut into 1 mm3 fragments and transplanted s.c. into the rightflanks of C57BL/6 mice using a trocar. When the tumorvolume reached 100mm3, the mice were randomly assigned intoa control group and a treatment group. The control group wasgiven isotype control rat IgG2a or IgG2b, and the treatment groupreceived either anti-mouse PD-1 InVivoMAb (clone RMP1-14,200 mg per mouse, Bio X Cell) or anti-mouse PD-L1InVivoMAb (clone 10F.9G2, 200 mg per mouse, Bio X Cell)intraperitoneally (i.p.) every 3 days for 11 to 15 days. The sizeof tumors was measured twice per week using a microcaliper.Tumor volume ¼ (length � width2)/2. Animal studies usingmouse models were approved by the Institutional Animal Careand Use Committee at Shanghai Institute of Materia Medica(approval no.2018-03-GMY-03).

Statistical analysisStatistical analysis was carried out with SPSS 17.0 (SPSS Inc.)

and Prism 6.0 (GraphPad). Quantitative data were presented asmeans� standard deviation (SD), except for mice tumor volumepresented as means � SEM. Comparisons between two groupswere performed using Student t test, Mann–Whitney U test, orWilcoxon rank-sum test. Survival data were analyzed by theKaplan–Meier method (log-rank test). Correlational analysis wasconducted using the c2 test. All tests were two-sided and a P valueof < 0.05 was considered statistically significant.

ResultsDownregulation of PD-L1 expression by FXR stratifies NSCLCpatients

First, we sought to confirm the negative correlation betweenFXR and PD-L1. For this, we extended the cohort of NSCLCspecimens with 408 cases in total to evaluate FXR and PD-L1expression by IHC staining. Due to their subcellular location fornormal functions, FXR nuclear staining and PD-L1 membranestaining in tumor cells were scored for further analysis (4, 17).Consistent with previous reports (18), PD-L1 expression wassignificantly higher in NSCLC than in pericarcinous lung tissues(Supplementary Fig. S1A and S1B). Moreover, PD-L1 wassignificantly lower in "FXR high" tumors than in "FXR low"tumors (Fig. 1A and B). c2 analysis revealed that there was astatistically significant inverse correlationbetween FXRandPD-L1in NSCLC samples (Table 1). We found that the proportion ofFXRhighPD-L1low tumors was up to 42.6% in all NSCLC samples.The survival curves for all four subgroups (FXRhighPD-L1low,FXRlowPD-L1high, FXRhighPD-L1high, and FXRlowPD-L1low) havebeen presented in Fig. 1C. Here, due to the inverse correlationbetween FXR and PD-L1 inNSCLC specimens, we focusedmainlyon the FXRhighPD-L1low and FXRlowPD-L1high subgroups andfound that the FXRhighPD-L1low subgroup had a significantlyworse overall survival as compared with FXRlowPD-L1high NSCLCpatients (Fig. 1C).

We asked whether the inverse correlation between FXR andmembrane PD-L1 in NSCLC specimens indicated a negativeregulatory interaction. We therefore measured the amount ofPD-L1 in FXR-silenced or -overexpressed NSCLC cells. SHP, adownstream gene target of FXR, was used to track alterations of

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FXR activity in experiments (19). Our data showed that FXRknockdown resulted in increased surface expression of PD-L1 inH1299, H1975, and HCC827 cells (Fig. 1D; SupplementaryFig. S2A and S2B). By contrast, stable FXR overexpression in

A549 and HCC4006 cells significantly suppressed cell-surfacePD-L1 expression compared with Mock groups (Fig. 1E; Supple-mentary Fig. S2C). Similar changes of PD-L1 mRNA were alsoobserved in these cells upon FXR silencing or overexpression(Fig. 1D and E; Supplementary Fig. S2A–S2C). We measuredexpression of PD-L1 in FXR-silenced or -overexpressed murinecancer cells as well. Accordingly, FXR knockdown in S91 signif-icantly increased PD-L1 surface expression andmRNA expression(Supplementary Fig. S3A), whereas stable FXR overexpression inLLC and MC38 cells significantly repressed PD-L1 expressioneither as protein at the cell surface or as mRNA (Fig. 1F; Supple-mentary Fig. S3B).

Figure 1.

The correlation between FXR and PD-L1 expressionin NSCLC. IHC staining was performed in 408NSCLC andmatched pericarcinous lung tissues.A, Representative images of FXRhighPD-L1low

staining and FXRlowPD-L1high staining in serialsections of NSCLC samples were shown at low(�30) and high (�200) magnifications (scale bar,50 mm). Negative control: without the primaryantibody. B, The IHC score of PD-L1 is lower in FXRhigh tumors (n¼ 215) than in FXR low tumors(n¼ 193; P¼ 0.0002, Mann–Whitney U test).C, Kaplan–Meier survival curves of FXRhighPD-L1low

(n¼ 139), FXRlowPD-L1high (n¼ 72),FXRhighPD-L1high (n¼ 37), and FXRlowPD-L1low

(n¼ 86) NSCLC patients (FXRhighPD-L1low vs.FXRlowPD-L1high, P¼ 0.014, log-rank test). D–F,H1299 cells (D) were transfected with NC- orFXR-siRNAs for 48 hours, and A549 (E) and LLC(F) cells were infected with lentiviral vectors toestablish mock or FXR-overexpressed stable celllines. Expression of FXR and SHP protein wasmeasured byWestern blot (left graphs),quantifications of mean fluorescence intensity(MFI) for PD-L1 surface staining were detected byflow cytometry (middle graphs), and relativeexpression of FXR, SHP, and PD-L1 mRNAwasmeasured by quantitative RT-PCR (right graphs).b-Actin was used as an internal control. Datarepresent mean� SD from at least threeindependent experiments. In D–F, P values weredetermined with the Student t test. � , P < 0.05,compared with the NC or Mock group, respectively.

Table 1. Correlation analysis between FXR and PD-L1 expression in NSCLCspecimens

PD-L1NSCLC samples Low High Correlation coefficient P value

FXR low 111 82 �0.255 <0.001a

FXR high 174 41aData were analyzed using the c2 test.






Molecular mechanisms by which FXR downregulates PD-L1expression in NSCLC

The molecular mechanisms by which FXR downregulatesPD-L1 expression in NSCLC were subsequently investigated. Asa transcription factor, FXR binds to FXRE in the DNA to regulatetranscription of target genes (20). Gene sequence analysisrevealed that the �1906/þ94 region of the human PD-L1 pro-moter contains a putative FXRE sequence (TGTTCAGTCACCT) atnucleotide�378 from the transcriptional initiation site.OurChIP

assays detected enriched bands from chromatin immunoprecipi-tated with anti-FXR in control H1975 andH1299 cells, comparedwith that of chromatin immunoprecipitated with isotype IgG(Fig. 2A). Moreover, FXR knockdown significantly abrogated therecruitment of FXR to the putative FXRE motif in H1975 andH1299 (Fig. 2A–C), whereas enforced FXR expression led tosignificant enhancement of FXR binding to PD-L1 promoter inHCC4006 cells (Fig. 2D and E). To expand upon these findings,luciferase reporter assays were carried out using pGL3 luciferase

Figure 2.

FXR downregulates PD-L1 expression in NSCLC viatransrepression and other mechanisms, includingSHP signals. ChIP assays were performed inH1975 and H1299 cells transfected with NC- orFXR-siRNAs, and FXR-overexpressed HCC4006stable cell lines by using anti-human FXR andprimers described in Materials and Methods.Chromatin obtained with isotype IgG andnon-immunoprecipitated samples (input) servedas a negative and positive control, respectively.A–E, Representative PCR amplification products inthe transfected H1975 (A, upper band) and H1299(A, lower band) cells, and HCC4006 stable cells(D) are shown, respectively. Enrichment of FXRprotein in the putative motif of PD-L1 promoter inthe transfected H1975 (B) and H1299 (C) cells, andHCC4006 stable cells (E) was determined relativeto input samples. F–H, FXR-silenced H1975 (F) andH1299 (G) cells, and FXR-overexpressed HCC4006stable cells (H) described above were transfectedwith a wild-type PD-L1 promoter plasmid(pGL3-PD-L1 FXRE-WT) or an FXRE-deleted PD-L1promoter plasmid (pGL3-PD-L1 FXRE-deleted), andthen collected for measuring luciferase activities24 hours after transfection. pGL3-basic plasmidserved as a negative control. I–J, H1299 (I) andFXR-overexpressed A549 stable cells (J) weretransfected with NC- or SHP-siRNAs for 48 hours.Left graphs, SHP and FXR protein expressiondetermined byWestern blot are shown. Rightgraphs, quantifications of MFI for PD-L1 surfacestaining are presented. Data represent mean� SDof three independent experiments. In B, C, E–J,P values were determined with Student t test. In B,C, and E, �, P < 0.05; �� , P < 0.01, compared withthe negative control; In F–H, � , P < 0.05, comparedwith FXRE-deleted PD-L1 promoter; in I–J,� , P < 0.05; �� , P < 0.01; ��, P < 0.001, comparedwith NC or Mock group, respectively; in B, C, E–H,and J, †, P < 0.05; ††, P < 0.01; †††, P < 0.001,compared with the corresponding NC or Mockgroup, respectively.

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constructs harboring the PD-L1 promoter with or without deletedFXRE element. In both H1975 and H1299 cells, FXR knockdownsignificantly increased the wild-type PD-L1 promoter activitywithout apparently affecting the activity of the FXRE-deletedPD-L1 promoter (Fig. 2F and G). In contrast, enforced FXRexpression significantly reduced the wild-type PD-L1 promoteractivity in HCC4006 cells, whereas the FXRE-deleted luciferasereporter was excluded from this reduction (Fig. 2H). The abovedata demonstrated that FXR can directly bind to the putative FXREmotif in PD-L1 promoter and repress its transcription.

We then asked if the effect of FXR on PD-L1 expressiondepends on its downstream effector, SHP (19). SHP silencingled to significantly increased PD-L1 expression in H1299 cells(Fig. 2I). SHP depletion reversed the downregulation of PD-L1induced by ectopic FXR overexpression in A549 cells (Fig. 2J),suggesting that SHP is involved in FXR-induced PD-L1 down-regulation in NSCLC cells.

In addition, we found that phosphorylated EGFR increased inEGFR-mutated FXR-silenced H1975 and HCC827 cells (Supple-mentary Fig. S4A and S4B), but not in EGFR-wild-type FXR-silencedH1299 or FXR-overexpressed A549 cells (SupplementaryFig. S4C). Subsequent experiments showed that afatinib andgefitinib suppressed this increase in phosphorylated EGFR, aswell as impaired the upregulation of PD-L1 either as protein at thecell surface or as mRNA induced by FXR knockdown in H1975and HCC827 cells (Supplementary Fig. S4A, S4B, S4D, and S4E),implying a functional role for EGFR signals in FXRsiRNA-inducedPD-L1 upregulation in EGFR-addicted NSCLC cells. Collectively,these results indicated that FXR downregulates PD-L1 via trans-repression and signaling through SHPandEGFR signals inNSCLCcells. On this basis, we defined the FXRhighPD-L1low NSCLCsubtype.

Thus, we found that FXR and PD-L1 expression were inverselycorrelated in NSCLC specimens. We identified a subgroup ofNSCLC patients harboring FXRhighPD-L1low tumor cells, in whichFXR downregulated PD-L1 expression.

FXRhighPD-L1low cells repress effector function andproliferation of CD8þ T cells

Next, we examined the effects of FXR on the tumor immunemicroenvironment in patients harboring FXRhighPD-L1low

NSCLC cells. Because CD8þ T cells represent the main target ofthe PD-L1/PD-1 checkpoint pathway in the TME (21), we usedin vitro coculture of FXRhighPD-L1low NSCLC cell lines with CD8þ

T cells or PBMCs or mouse splenocytes. We tested CD8þ T-cellfunction and proliferation. In addition to the depressed PD-L1expression in FXR-overexpressingA549 cells, cells coculturedwithpurified CD8þ T cells induced a decreased ratio of activated CD8þ

T cells to total CD8þ T cells, evident from a decrease in CD8þ Tcells positive for either IFNg or TNFa compared with when CD8þ

T cells were cocultured with mock A549 cells (Fig. 3A; Supple-mentary Fig. S5A). Similar results were obtained from PBMCscocultured with FXR-overexpressing A549 cells (Fig. 3B). Underthe same coculture conditions, CD8þ T-cell proliferation wasinhibited upon FXR high expression (Supplementary Fig. S5B).We observed similar effects of FXR in the FXRhighPD-L1low mousecancer cell line. Therewas a nonsignificant trend toward a reducedratio of IFNgþ CD8þ T cells or TNFaþ CD8þ T cells followingcoculture of mouse CD8þ T cells with FXR-overexpressing LLCcells as compared with the mock LLC control (Fig. 3C; Supple-mentary Fig. S5C). Accordingly, the proportion of CD8þ T cells

Figure 3.

FXR represses cocultured CD8þ T-cell functions in FXRhighPD-L1low NSCLCcells. A and B, Purified human CD8þ T cells from PBMCs (A) or human PBMCs(B) were cocultured with A549 stable cells overexpressing FXR or not for 3days. C and D, Purified mouse CD8þ T cells from splenocytes (C) or mousesplenocytes (D) were cocultured with LLC stable cells overexpressing FXR ornot for 3 days. Intracellular staining for IFNg or TNFa in human andmouseCD8þ T-cell subpopulations was measured by flow cytometry. Datarepresent mean� SD of three independent experiments. P values weredetermined with Student t test. � , P < 0.05; �� , P < 0.001, compared withthe Mock group. ns, not significant.






positive for either IFNg or TNFawas significantly decreased whenmouse splenocytes were cocultured with FXR-overexpressed LLCcells instead of themock cells (Fig. 3D). These data showed that inFXRhighPD-L1low NSCLC cells, FXR represses antitumor CD8þ

T-cell proliferation and function in vitro.

FXR constructs an immunosuppressive microenvironment inmouse models

To determine whether the immunosuppressive effects of FXRin FXRhighPD-L1low NSCLC cells could be recapitulated in vivo,C57BL/6 mice were subcutaneously inoculated with LLC cellswith or without ectopic FXR, and the tumor-infiltratingimmune cells were characterized. Consistent with our previousstudy (11), enforced FXR expression in LLC led to a significantlyincreased tumor growth in this murine syngeneic tumor model(Fig. 4A). IHC analysis demonstrated downregulation ofmembrane PD-L1 in mouse LLC-FXR tumors, indicating anFXRhighPD-L1low phenotype, as compared with LLC-Mocktumors (Fig. 4B). Results showed that although the frequencyof total hematopoietic cells (CD45þ) did not differ between thetwo groups, LLC-FXR tumors exhibited significantly increasedinfiltration of myeloid cell populations, such as dendritic cells(DCs) and myeloid-derived suppressor cells (MDSCs), andsignificantly decreased infiltration of lymphoid cell popula-tions, such as CD8þ cytotoxic T cells, natural killer (NK) cells,and CD4þ effector T cells, as compared with LLC-Mock tumors(Fig. 4C). We found a lower ratio of CD8þ T cells to regulatoryT cells (Treg) and decreased DC activation in the LLC-FXRgroup as compared with the LLC-Mock group (Fig. 4C). Wefurther interrogated molecular features of these immune cellsand found significantly increased PD-1 and increased, but notsignificant, exhaustion marker Lag-3 expression on NK cells inLLC-FXR tumors than in LLC-Mock tumors (Fig. 4C). CD8þ Tcells from FXR-overexpressed tumors exhibited an inactivatedand exhausted phenotype, shown as significantly decreasedTNFaþ CD8þ T cells as well as increased Lag-3 expression onCD8þ T cells relative to mock tumors (Fig. 4C). These findingswere consistent with our observations in vitro, showing that FXRsuppresses tumor-infiltrating immune cells and thus constructsan immunosuppressive microenvironment in FXRhighPD-L1low

mouse LLC tumors.

Anti–PD-1 immunotherapy is effective against FXRhighPD-L1low

mouse LLC tumorsWe then sought translational implications for the

FXRhighPDL1low NSCLC subtype. Thus, anti–PD-1 was used.Anti–PD-L1 was included as a NC. Our results showed thatanti–PD-1 led to significant tumor inhibition in FXR-overex-pressed LLC tumors, but caused almost no change in mock LLCtumors (Fig. 5A). In parallel, anti–PD-L1 induced no significantchange in terms of tumor volume in mouse LLC-FXR tumors(Supplementary Fig. S6A).

The tumor-infiltrating immune cells were then analyzed usingflow cytometry. We found that the frequency of T cells expressingCD69, IFNg , or TNFa was significantly higher in anti–PD-1-treated LLC-FXR tumors than in control LLC-FXR tumors(Fig. 5B). In contrast, the infiltration of Tregs within mouseLLC-FXR tumors was significantly reduced by PD-1 blockade(Fig. 5C). As expected, the frequency of T cells expressing CD69,IFNg , TNFa, or Granzyme B did not differ significantly betweenanti–PD-L1-treated LLC-FXR tumors and control tumors, even

though decreased Treg infiltration was observed within LLC-FXRtumors treated with anti–PD-L1 (Supplementary Fig. S6B andS6C). In total, thesefindings demonstrated that anti–PD-1 immu-notherapy was effective against FXRhighPD-L1low mouse LLCtumors, which were associated with a reactivation of antitumorimmunity.

DiscussionThe TME, especially its immunologic attributes, plays a role in

NSCLC progression (3). FXR has been implicated as a regulatorof inflammation and immune responses in various dis-eases (12, 13). Zhang and colleagues found a protective andanti-inflammatory role of FXR in a lipopolysaccharide-inducedmousemodel of acute lung injury (14). But the relevanceof FXR tothe immune microenvironment of NSCLC has been unclear.Here, we found an inverse correlation between FXR and PD-L1expression in a cohort of 408 samples from patients with NSCLC.We identified an FXRhighPD-L1low NSCLC subgroup. Indeed,PD-L1 was downregulated by FXR in NSCLC cells. We showedin vitro and in an animal model that FXR constructs an immu-nosuppressive microenvironment, characterized by inactivatedand exhausted CD8þ T cells, that enables anti–PD-1 therapyin FXRhighPD-L1lowmouse LLC tumors. The present study extendsFXR function to an immunosuppressive role in FXRhighPD-L1low

NSCLC subtype, and recommends FXRhighPD-L1low as abiomarker to guide anti–PD-1 immunotherapy.

We observed an inverse correlation between FXR and PD-L1 inNSCLC samples, pointing to a subgroup of FXRhighPD-L1low

NSCLC patients. In support of this definition, we found that FXRknockdown led to increased PD-L1 expression, whereas FXRoverexpression induced PD-L1 downregulation either in NSCLCcells or in murine cancer cells. These results, which characterize afunctional role for FXR in PD-L1 regulation in cancer, are con-sistent with suggestions from a previous study, showing that FXRagonists' regimen could reduce the expression of PD-L1 and PD-1in spleen CD4þ T cells and CD19þ B cells in an experimentalautoimmune encephalomyelitis mouse model (22). Mechanisti-cally, our data showed that FXR can repress PD-L1 transcription bybinding to the putative FXRE element in the PD-L1 promoter. Inaddition, SHP and EGFR signals are involved in FXR-inducedPD-L1 downregulation in NSCLC cells.

We previously found that FXR contributes to tumor growththrough increasing cyclin D1 transcription in NSCLC (11). In thisstudy, the tumor-promoting effect of FXR was verified in animmune-competent murine LLC tumor model. FXR was foundto repress effector function and proliferation of CD8þ T cells,which were cocultured in vitrowith FXRhighPD-L1low NSCLC cells.In the context of FXRhighPD-L1low mouse LLC tumors, FXR remo-deled an immunosuppressive microenvironment, as manifestedby increased infiltration of myeloid cell populations, such asMDSCs, and decreased infiltration of antitumor lymphoid cellpopulations, such as CD8þ cytotoxic T cells, NK cells, and CD4þ

effector T cells. CD8þ T cells from FXR-overexpressed tumorsexhibited an inactivated and exhausted phenotype (shown asdecreased TNFaþCD8þ T cells as well as increased Lag-3 expres-sion on CD8þ T cells). These results are in accordance with priorstudies showing the requirement for FXR activation in inducinganti-inflammatory macrophages and MDSCs as well as in sup-pressing T lymphocyte responses in mouse models of autoim-mune diseases (22–24). Based on the change ofmultiple immune

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cells in FXR-overexpressed LLC tumors, we reasoned that FXRshould influence different cell types within the tumor immunemicroenvironment. In agreementwith this notion, our in vivo data

showed that forced expression of FXR in mouse LLC tumorscontributed to decreased PD-L1 expression, not only on tumorcells but also on infiltrating immune cells. However, our study has

Figure 4.

FXR constructs an immunosuppressive microenvironment in FXRhighPD-L1low mouse LLC tumor models. C57BL/6mice were inoculated s.c. with 1� 106

LLC-Mock or LLC-FXR cells. A, Comparison of tumor growth between LLC-Mock and LLC-FXRmouse tumor models (n¼ 7). The tumor volume was shown onindicated days as the mean� SEM. B, Representative IHC images for FXR and PD-L1 staining in serial sections of LLC-Mock or LLC-FXR tumors are shown(scale bar, 50 mm). Negative control: without the primary antibody. C, Flow-cytometric analysis of immune subsets among CD45þ cells in LLC-Mock (n¼ 6)and LLC-FXR (n¼ 8) mouse tumors. GzmB, Granzyme B. In A and C, P values were determined with the Student t test, respectively. �, P < 0.05; �� , P < 0.01;�� , P < 0.001 compared with the mock group. ns, not significant.






some limitations. The detailed mechanisms for FXR-modulatedimmunosuppressive microenvironment in FXRhighPD-L1low

NSCLC subtype have not been illustrated in the present work.Further studies are warranted to uncover it.

Immunotherapy has changed the landscape of lung cancertreatment. Immune-checkpoint inhibitors, especially for PD-L1/PD-1-blocking antibodies, have improved treatments forpatients with advanced NSCLC (25–27). Preliminary studiessuggested that PD-L1 expression in tumors is often associatedwith a higher likelihood of response to PD-L1/PD-1 inhibi-tors (28). However, some patients with low or negative PD-L1expression couldalsobenefit fromPD-L1/PD-1blockade (26,29).

Our in vivo treatment experiments showed an increased suscep-tibility to anti–PD-1 in FXRhighPD-L1low mouse LLC tumors thanin mock LLC tumors. Given the relatively intact antitumorimmune responses preexisting in mock tumors, the impairedimmune milieu, especially the exhausted CD8þ T phenotype,is likely to rendermousemodelsmore susceptible to anti–PD-1 inFXR-overexpressed LLC tumors, because it is readily reactivatedafter immune-checkpoint inhibition. In addition to tumor reduc-tion, we detected increased proportions of CD69þ, IFNgþ, orTNFaþCD8þ T cells in FXR-overexpressed tumors after anti–PD-1 treatments, suggesting a restoration of antitumor immunity.On the other hand, anti–PD-L1 was ineffective in treating

Figure 5.

Anti–PD-1 therapy is effective againstFXRhighPD-L1low mouse LLC tumors. C57BL/6mice were inoculated s.c. with 1� 106

LLC-Mock or LLC-FXR cells, andadministrated i.p. with 200 mg anti-mousePD-1 or corresponding isotype control IgG2aevery 3 days for 13 or 15 days after the tumorvolume reached 100mm3.A, Comparisonsof tumor growth in LLC-Mock (n¼ 8) orLLC-FXR (n¼ 7) tumors treated withanti–PD-1 versus isotype IgG2a. B and C,Flow-cytometric analysis of CD8þ T cellspositive for CD69, IFNg , TNFa, or GzmB (B),or Foxp3high Tregs (C) in mouse LLC-FXRtumors (n¼ 7) treated with anti–PD-1 orisotype IgG2a. P values were determinedwith the Student t test. � , P < 0.05;�� , P < 0.01, compared with isotypecontrol IgG2a. ns, not significant.

You et al.





FXRhighPD-L1low mouse LLC tumors, perhaps due to the absenceof targetable PD-L1 on tumor cells. The tumor immune micro-environment determines the response to anti–PD-1/anti–PD-L1immunotherapy (30, 31). Our findings support FXRhigh as asensitive biomarker for anti–PD-1 immunotherapy in PD-L1low/negative NSCLC patients. Further pilot studies ofFXRhighPD-L1low NSCLC specimens treated with anti–PD-1 areneeded to verify the value of FXRhigh status in predicting immu-notherapy responses when PD-L1 is low. Due to the immuno-suppressive status of the TME (characterized by increased CD206on macrophages, decreased MHC II and CD86 on DCs, andincreased Lag-3 on CD8þ T cells in FXR-overexpressed LLCtumors), we propose that the FXRhighPD-L1low NSCLC patientsmight be cured by other immunotherapies, such as tumor-associated macrophage blockade or DC-based therapy, as previ-ously reported (32, 33). Further studies are needed to analyze thishypothesis.

In summary, we have identified a subgroup of NSCLC patientscharacterized by FXRhighPD-L1low tumor cells, in which FXRdownregulated PD-L1 expression. Our findings have uncoveredan immunosuppressive role for FXR in the FXRhighPD-L1low

NSCLC subtype. Our study provides translational insights intothe therapeutic activity in PD-L1low NSCLC patients treated withanti–PD-1. We suggest FXRhighPD-L1low be considered as a bio-marker for anti–PD-1 immunotherapy in the future.

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

Authors' ContributionsConception and design: J. Ai, H. JiangDevelopment of methodology: X. Liu, S. Xue, B. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. You, L. Li, D. Sun, Z. XiaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. You, L. Li, D. Sun, Z. Xia, J. Ai, H. JiangWriting, review, and/or revision of the manuscript: W. You, J. Ai, H. JiangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Chen, H. QinStudy supervision: J. Ai, H. Jiang

AcknowledgmentsThis work was supported in part by grants 91629104, 81874314, 81821005,

and 81773762 from the National Natural Science Foundation of China andgrants XDA12020000, XDA12020103 from the Personalized Medicines -Molecular Signature-based Drug Discovery and Development Strategic PriorityResearch Program of the Chinese Academy of Sciences.

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 22, 2017; revised July 14, 2018; accepted April 5, 2019;published first April 11, 2019.

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You et al.




2019;7:990-1000. Published OnlineFirst April 11, 2019.Cancer Immunol Res Wenjie You, Lijun Li, Deqiao Sun, et al.

PD-1 Immunotherapy−Anti NSCLC to lowPD-L1highMicroenvironment and Sensitizes FXR

Farnesoid X Receptor Constructs an Immunosuppressive

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