ctla4 promotes tyk2-stat3 dependent b-cell oncogenicity...duarte, california. 5hematology institute,...

Microenvironment and Immunology

CTLA4 Promotes Tyk2-STAT3–Dependent B-cellOncogenicityAndreas Herrmann1, Christoph Lahtz1, Toshikage Nagao1,2, Joo Y. Song3,Wing C. Chan3, Heehyoung Lee1, Chanyu Yue1, Thomas Look1, Ronja M€ulfarth1,Wenzhao Li1, Kurt Jenkins4, John Williams4, Lihua E. Budde5, Stephen Forman5,Larry Kwak5, Thomas Blankenstein6, and Hua Yu1

Abstract

CTL–associated antigen 4 (CTLA4) is a well-establishedimmune checkpoint for antitumor immune responses. The pro-tumorigenic function of CTLA4 is believed to be limited to T-cellinhibition by countering the activity of the T-cell costimulatingreceptor CD28. However, as we demonstrate here, there are twoadditional roles for CTLA4 in cancer, including via CTLA4 over-expression in diverse B-cell lymphomas and in melanoma-associated B cells. CTLA4-CD86 ligation recruited and activatedthe JAK family member Tyk2, resulting in STAT3 activation andexpression of genes critical for cancer immunosuppressionand tumor growth and survival. CTLA4 activation resulted in

lymphoma cell proliferation and tumor growth,whereas silencingor antibody-blockade ofCTLA4 inB-cell lymphoma tumor cells inthe absence of T cells inhibits tumor growth. This inhibition wasaccompanied by reduction of Tyk2/STAT3 activity, tumor cellproliferation, and induction of tumor cell apoptosis. The CTLA4–Tyk2–STAT3 signal pathway was also active in tumor-associatednonmalignant B cells in mouse models of melanoma and lym-phoma. Overall, our results show how CTLA4-induced immunesuppression occurs primarily via an intrinsic STAT3 pathway andthat CTLA4 is critical for B-cell lymphoma proliferation andsurvival. Cancer Res; 77(18); 5118–28. �2017 AACR.

IntroductionCTL–associated antigen 4 (CTLA4) is well recognized as an

immune checkpoint, and has emerged as a prominent target forcancer immunotherapy (1, 2). CTLA4-blocking antibodies, alongwithPD1andPD-L1–blocking antibodies, are capable of unleash-ing antitumor immune responses with durable cancer regression(1, 2). However, despite being one of the most potent anticancerdrugs, CTLA4-blocking antibodies are unable to significantlyprolong the lives of majority of the treated patients, suggestingan urgent need to further understand CTLA4 biology in cancer,thereby enabling the development of rational combinatory

approaches to optimize the antitumor efficacy of CTLA4-blockingantibodies.

Themechanism by which CTLA4 dampens T-cell responses hasbeen attributed to the fact that CTLA4 shares identical ligands,B7-1 (CD80)/B7.2 (CD86; refs. 3, 4) on antigen-presenting cells,with T-cell costimulating receptor CD28. However, whether andhow CTLA4 may dampen T-cell activation through cell-intrinsicmechanism remains unknown. In addition, although it is con-sidered expressed exclusively by T cells, there are some indicationsthat CTLA4 is expressed by certain malignant B cells (5). If CTLA4is consistently and highly expressed by B cells in the tumormicroenvironment, it would suggest that B cells could also damp-en T-cell activation by competing with CD28 for engaging B7-1(CD80)/B7.2 (CD86) on antigen-presenting cells. However, theseconcepts have not been formerly tested.

A critical role of tumor-associated B cells in promoting cancersurvival/resistance to therapies aswell as immunosuppressionhasbeen reported (6–13). Among several mechanisms, STAT3 hasbeen shown to mediate the cancer promoting activities of tumor-associated B cells (12, 13). STAT3 is persistently activated indiverse cancers, including many B-cell malignancies (14, 15).STAT3 is critical for upregulating the expression of numerousgenes involved in cancer cell survival/proliferation, and invasion(16). A standout feature of STAT3 in cancer is that it also promotesexpression of an array of immunosuppressive genes while inhi-biting many Th1 immunostimulatory genes necessary for, induc-ing antitumor T-cell immunity (16–18). STAT3 activity in malig-nant B cells has been shown to inhibit the antigen presentationability of these cells (19). STAT3 is persistently activated in diverseimmune subsets in the tumormicroenvironment, includingmye-loid cells, B cells, as well as T-cell, inducing immunosuppressionand promoting tumor growth (4, 12–15, 20). Nevertheless, the

1Department of Onco-Immunology, Beckman Research Institute, City of HopeComprehensive Cancer Center, Duarte, California. 2Department of Hematology,Graduate School of Medical and Dental Science, Tokyo Medical and DentalUniversity, Tokyo, Japan. 3Department of Pathology, City of Hope Comprehen-sive Cancer Center, Duarte, California. 4Department of Molecular Medicine,Beckman Research Institute, City of Hope Comprehensive Cancer Center,Duarte, California. 5Hematology Institute, City of Hope Comprehensive CancerCenter, Duarte, California. 6Max-Delbr€uck-Center for Molecular Medicine, andthe Institute of Immunology, Charit�e Campus Buch, Berlin, Germany.

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

A. Herrmann, C. Lahtz, and T. Nagao contributed equally to this article.

Corresponding Authors: Andreas Herrmann, Beckman Research Institute—Cityof Hope, Beckman Building, 1500 East Duarte Road, Duarte, CA 91010-3000.Phone: 626-256-4374, ext. 64428; Fax: 626-256-8708; E-mail:[email protected]; and Hua Yu, [email protected]

doi: 10.1158/0008-5472.CAN-16-0342

�2017 American Association for Cancer Research.

CancerResearch

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upstreammolecules/receptors that activate STAT3 in malignant Bcells and in tumor-associated "normal" B and T cells remain to befurther explored. In this study, we investigated the potential roleof CTLA4 in B cells in promoting tumor progression. Our studiesidentified a cell-intrinsic immunosuppressive pathway for CTLA4and an unexpected function of CTLA4 in promoting tumor cellgrowth and survival.

Materials and MethodsMice and cell culture

For subcutaneous tumor challenge, C57BL/6, Balb/c (TheJackson Laboratory) or athymic nude mice (NCI Frederick), wereinjected with 105 B16 melanoma or 2.5 � 105 A20 lymphoma,respectively. Athymic nu/nu mice (NCI Frederick) were engraftedwith 2� 106 Ly3 human lymphoma cells subcutaneously into theflank. After tumors reached 5 to 7mm in diameter, treatmentwith250 mg/dose/mouse CTLA4 blocking antibody (BioXCell) waslocally administered every other day.

Human B-cell lymphoma Ly3, U266 cells (kindly provided in2010 by Dr. Ana Scuto, Beckman Research Institute at the Com-prehensiveCancer Center at theCity ofHope,Duarte, CA),Daudi,JeKo-1, SU-DHL-6, Raji and RPMI6666 cells (ATCC obtained in2016) were cultured in IMDM or RPMI medium (Gibco), respec-tively, human multiple myeloma MM.1S (kindly provided in2016 by Dr. Stephen Forman, Comprehensive Cancer Center atthe City of Hope) and H929 (ATCC) were cultured in DMEMmedium supplemented with 10% FBS (Sigma) and 0.05 mol/Lmercaptoethanol. Mouse DC2.4 dendritic cells (kindly providedin 2008 by Dr. Marcin Kortylewski, Beckman Research Institute atthe Comprehensive Cancer Center at the City of Hope), A20 B celllymphoma (ATCC obtained in 2009), andmouse B16melanoma(kindly provided in 2007 by Dr. Drew Pardoll, The SidneyKimmel Comprehensive Cancer Center at Johns Hopkins Schoolof Medicine, Baltimore, MD) were grown in RPMI1640 (Gibco)containing 10% FBS. Mouse RAW264.7 macrophages (ATCC,obtained in 2010) were cultured in DMEM supplemented with10% FBS. Cells used in this study were routinely freshly thawed,subcultured for up to 3 weeks for desired in vitro studies or in vivoengraftment, tested for mycoplasma contamination and authen-ticated by RT-PCR and flow cytometry. Cell subculture wasimmediately amplified for long-term storage in liquid nitrogen.

Study approvalMouse care and experimental procedures with mice were per-

formed under pathogen-free conditions in accordance with estab-lished institutional guidance and approved Institutional AnimalCare and Use Committee protocols from the Research AnimalCare Committees of the City of Hope.

Patient tumor specimensThis study was performed in accordance with the Helsinki

principles and approved by the Institutional Review Board atCity of Hope Medical Center (IRB14225). Informed writtenconsent was obtained. The human tumor samples were evaluatedby physicians at Department of Pathology of City of Hope.Detailed information is summarized in Tables 1 and 2.

Generating stable cell linesTo generate BA/F3 cell lines stably expressing human CTLA4

constructs, murine pro-B-cell line BA/F3 was grown in IL3 con-

taining RPMI1640medium containing 10%FBS, 10 ng/mL IL3 or10% conditionedmediumofWEHI-3B cell line. MouseWEHI-3Bcells were grown in Iscove's MDM supplemented with 5% to 10%FBS, 2mmol/L L-glutamine, and 2.5� 10�5mol/Lmercaptoetha-nol. Human CTLA-GFP constructs were introduced by electropo-ration. Briefly, 3.5 � 106 BA/F3 cells were resuspended in 800 mLcell culturemedia containing 28 mg vector. Cells were pulsed with200 V for 70 msec and subcultured.

Human B-cell lymphoma Ly3 cells with knocked downhumanCTLA4 expressionwere generated using lentiviral shRNAparticles obtained from Santa Cruz Biotechnology. Cellularintroduction of shRNA was carried out according to the manu-facturer's instructions.

PlasmidsPlasmid coding for mouse CD86-mCherry was obtained from

(GeneCopoeia). Plasmid encoding human CTLA4-GFP was pur-chased fromOriGene (RG210150). Site directedmutagenesis wasperformed using QuickChange (Stratagene), resulting in hCTLA4constructs hCTLA4-Y201F (50-ctcttacaacaggggtctttgtgaaaatgccccca-30; 50-tgggggcattttcacaaagacccctgttgtaagag-30) and Y218F (50-gcaatttcagcctttttttattcccatcaatacgcgtacg-30; 50-cgtacgcgtattgatgggaa-taaaaaaaggctgaaattgc-30).

Generation of soluble human CD86Human CD86 gene was obtained fromDNASU plasmid repos-

itory (clone: HsCD00039473). Soluble human CD86-Fc gene inpVL1393 vector was transfected into Sf9 cells with BestBac 2.0Baculovirus Cotransfection kit (Expression Systems). High titer

Table 1. Human diffuse large B-cell lymphoma/NHL tumor samples (IRB14225)

Diffuse large B-cell lymphoma, DLBCLSample Diagnosis Site Age Sex

1 DLBCL Lymph node 72 M2 DLBCL Lymph node 60 M3 DLBCL Lymph node 77 M4 DLBCL Lymph node 74 M5 DLBCL Soft tissue 51 M6 DLBCL Lymph node 58 F7 DLBCL GI 39 F8 DLBCL Lymph node 71 M9 DLBCL Lymph node 32 F10 DLBCL Lymph node 19 M11 DLBCL Lymph node 72 F

NOTE: The human tumor samples included in this study were evaluated byphysicians at Department of Pathology of City of Hope.

Table 2. Human follicular lymphoma/NHL tumor samples (IRB14225)

Follicular lymphoma, FLSample Diagnosis Site Age Sex

1 FL1-2 Lymph node 58 F2 FL3A Lymph node 62 M3 FL3A Lymph node 76 M4 FL1-2 Lymph node 72 F5 FL1-2 Lymph node 71 M6 FL3A Lymph node 61 M7 FL3A Lymph node 66 F8 FL3A Lymph node 61 F9 FL3A Lymph node 39 M10 FL1-2 Lymph node 65 M11 FL3A Lymph node 55 M

NOTE: The human tumor samples included in this study were evaluated byphysicians at Department of Pathology of City of Hope.

CTLA4 in B Cells Promotes Oncogenicity via Tyk2-STAT3

www.aacrjournals.org Cancer Res; 77(18) September 15, 2017 5119



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virus was generated and used to infect Tni cells at an MOI of 3 forprotein production. Cells were harvested 48 hours postinfection,centrifuged at 4,000 rpm for 25 minutes, and the filtered super-natant was applied to a Protein A resin (GenScript). After PBSwash, protein was eluted with 0.1 mol/L glycine, pH 3.0 andimmediately pH adjusted with 1 mol/L Tris-HCl pH 8.0. Con-centrated eluate was applied to HiLoad 26/60 Superdex 200column(GEHealthcare) inPBS. Peak fractionswere concentrated,flash frozen, and stored at �80� C. Purity was monitored bySDS-PAGE.

Generated and purified human sCD86 was fluorescentlylabeled. Briefly, peptide diluted in 200-mL PBS was activated witha 1:10 dilution of 1mol/LNaHCO3 (20mL),mixedwith a grain ofNHS coupled AlexaFluor 647 (Invitrogen) dissolved in 2 mLDMSO (Sigma), and incubated light protected at room temper-ature for 1 hour up to 1.5 hours. Gel filtration columnwas packedwith G75 Sephadex (GE Healthcare) and fluorescently labeledsCD86 peptide was eluted by centrifugation for 5 minutes at1,100 � g.

ImagingIndirect immunoflourescence and IHC were carried out as

described previously (19) staining CD3, CD20 (BioLegend),CTLA4, c-Myc, pSTAT3 (Santa Cruz Biotechnology), Hoechst33342 (Sigma), Ki67 (Vector), CD19, CD31 (BioLegend, BDBiosciences), pTyk2 and cleaved caspase-3 (Cell Signaling Tech-nology). CFSE was purchased from Invitrogen and CFSE loadinginto cells was carried out according to the manufacturer's instruc-tions. Imaging was carried out on a confocal microscope ZeissLSM510 Meta.

Flow cytometryCell suspensions isolated from tissue were prepared as

described previously (20) and stained with different combina-tions of fluorophore-coupled antibodies to CD3, CD4, CD8,CD19, CD28, CD62L, CD69, CD80, CD86, B220, CTLA4, phos-pho-Tyr705-Stat3, FoxP3, IFNg , IL4 (BD Biosciences). Antibodiesagainst c-Myc and pTyk2 were purchased from Cell SignalingTechnology; staining was performed using a fluorescently labeledsecondary antibody (Invitrogen). Fluorescence data were collect-ed on Accuri or Fortessa flow cytometers (BD Biosciences) andanalyzed using FlowJo software (Tree Star).

Immunoblotting, immunoprecipitationWhole cell lysates were prepared using RIPA lysis buffer

containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl,1 mmol/L EDTA, 0.5% NP-40, 1 mmol/L NaF, 15% glycerol,and 20 mmol/L b-glycerophosphate. A protease inhibitor cock-tail was added fresh to the lysis buffer (Mini Protease InhibitorCocktail, Roche). Normalized protein amounts were subjectedto electrophoretic separation by SDS-PAGE, transferred ontonitrocellulose for Western blotting, and subsequently immu-nodetection was performed using antibodies against STAT3,Tyk2, PY99 (Santa Cruz Biotechnology), anti-pTyr (clone4G10, Millipore) and b-actin (Sigma). For coimmunoprecipi-tation, CTLA4, JAK1, JAK2, JAK3, Tyk2 antibodies (Santa CruzBiotechnology) were used to label rProtein G agarose beads(Invitrogen), subsequently incubated for 16 hours with whole-cell lysates, subjected to electrophoretic protein separation andWestern blot detection.

Electrophoretic mobility shift assayNuclear extracts from cells were isolated using buffer A contain-

ing 10 mmol/L HEPES/KOH pH 7.9, 1.5 mmol/L MgCl2,10mmol/L KCl and buffer C containing 20mmol/LHEPES/KOHpH 7.9, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/LEDTA, 25% glycerol; per 2 mL buffer, protease inhibitors at0.2 mmol/L PMSF, 0.5 mmol/L DTT, and 1 mmol/L Na3VO4were added fresh before use. Cells were washed with PBS, resus-pended in buffer A, incubated on ice for 20 minutes and sedi-mentedby centrifugation for 20 seconds at 13.2 rpm in a table-topcentrifuge. Pellet was resuspended in buffer C, incubated for30 minutes on ice and sedimented by centrifugation for 10 min-utes at 13.2 rpm. Double-stranded DNA SIE oligo (50-AGCTT-CATTTCCCGTAAATCCCTA-30/30AGTAAAGGGCATTTAGGGAT-TCGA-50 containing STAT1and STAT3 consensus binding site wasradiolabeled with 32P-ATP/32P-CTP using Klenow enzyme (Pro-mega). Nuclear extracts were resuspended at 10 mg with loadingbuffer (50 mmol/L HEPES pH 7.8, 5 mmol/L EDTA pH 8,25 mmol/L MgCl2 adjusted to pH 7.8 with 3 mol/L KOH)containing radiolabeled SIE-oligo and separated by PAGE elec-trophoresis; dried gel was exposed on X-ray film to assess STAT3DNA binding. For supershift analysis, aSTAT3 antibody (C-20X,Santa Cruz Biotechnology) was added to nuclear extract at1 mL/20 mL and incubated on ice for 15 minutes before loadingonto PAGE for electrophoretic separation.

PCRTranscript amplification was determined from total RNA puri-

fied using the RNeasy Kit (Qiagen). cDNA was synthesized usingthe iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR wasperformed in triplicates using the Chromo4 Real-Time Detector(Bio-Rad). The humanGAPDHhousekeeping genewas used as aninternal control to normalize target gene mRNA levels. Primerswere obtained from SA Biosciences (human BCL2L1:PPH00082B-200, human MMP9: PPH00152E-200) or custom-ized from Integrated DNA Technologies IDT (human IL6: hIL6 F:50-GTACATCCTCGACGGCATC-30, R: 50-CCTCTTTGCTGCTTT-CACAC-30, human IL10: hIL10 F: 50-TGCCTAACATGCTTCGA-GATC-30, R: 50-GTTGTCCAGCTGATCCTTCA-30, human IFNg:hINFG F: 50-GAGATGACTTCGAAAAGCTGAC-30, R: 50-CACTTG-GATGAGTTCATGT ATTGC-30).

Statistical analysisStatistical analyses were performed using Prism (GraphPad)

software. The overall significance for each graph was calculatedusing the two-tailed Student t test. P values of less than 0.05 wereconsidered statistically significant.

ResultsMalignant B cells express functional CTLA4

To date, CTLA4 regulatory functions are considered only in Tcells (2). However, it has been suggested that CTLA4 is alsoexpressed in certain malignant B cells (5). We therefore assessedCTLA4 expression in patient B-cell lymphoma biopsies. Weobserved considerably elevated CTLA4 expression by tumor infil-trating CD3þ T cells as well as in CD20þ cells in human B-celllymphoma tissues (Fig. 1A, top). Compared with normal lymphnode, expression of CTLA4 is significantly increased in lymphnode with B-cell lymphoma (Fig. 1A, bottom). We also assessedCTLA4 expression in twomain types of humanNHL lymphomas,

Herrmann et al.

Cancer Res; 77(18) September 15, 2017 Cancer Research5120




A CD3 CTLA4 Merge CD20 CTLA4 Merge

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Figure 1.

CTLA4 expression and function by B-cell lymphoma cells. A, IHC staining followed by confocal microscopy analyses showing CTLA4 expressionin CD3þ T cells and CD20þ cells in human B-cell lymphoma tissues. Indicated areas (white boxes) are magnified; scale, 50 mm (top). CTLA4 expression in normalhuman lymph node versus lymph node with B-cell lymphoma, shown by confocal images and quantification (bottom). SD shown; t-test: �� , P < 0.001. B,Representative microscopic images showing elevated CTLA4 expression by human B-cell lymphoma DLBCL and FL (left) tumor sections. Quantified frequency ofCTLA4 expression in all of the analyzed patient tumor biopsies (n ¼ 11 for both tumor types; right); scale, 50 mm. C, CTLA4 surface expression by human B-celllymphoma cell line Ly3 assessed by flow cytometry. Flow cytometry (D) and confocal microscopy (E) showing cellular internalization of soluble CD86 by Ly3 cells;scale bar, 10 mm.






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Figure 2.

CTLA4 contributes to CD86 cellular internalization. A, CTLA4-positive A20 B-cell lymphoma cells uptake CD86 from APCs. CD86-mCherry–expressingRAWmacrophages were cocultured with CFSEþA20 cells. Cellular internalization of full-length CD86-mCherry by A20 cells was visualized by confocal microscopy;scale, 10 mm. B, Flow cytometric quantitative analysis showing CD86-mCherry cellular internalization expressed by RAW macrophages (top) or dendriticcells (bottom) by CFSEþA20 cells. C, Flow cytometric analyses of CD80, CD86, CD28, and CTLA4 in murine A20 B-cell lymphoma cells.D, CTLA4 blockade reducessCD86 internalization by human B-cell lymphoma Ly3 (top) and CTLA4þ Raji (bottom) cells assessed by flow cytometry.

Herrmann et al.





diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma(FL; Tables 1 and 2). We show that CTLA4 is detectable in bothtypes of NHL lymphomas (DLBCL, 81% and FL, 36%; Fig. 1B).

CTLA4 is also expressed in tested cell lines derived fromhuman B malignancies, including Ly3 (DLBCL; Fig. 1C; Sup-plementary Fig. S1A and S1B) and human multiple myelomacell lines (Supplementary Fig. S1). CTLA4þ B-cell lymphomacells rapidly engaged with soluble CD86 (sCD86; Fig. 1D;Supplementary Fig. S1C), allowing CD86 cellular internaliza-tion (Fig. 1E). Incubating murine RAW macrophages expres-sing fluorescently labeled full-length CD86-mCherry withmouse B-cell lymphoma A20 cells loaded with CFSE resultedin a CD86-mCherryþ A20 B-cell lymphoma population, asshown by confocal microscopy (Fig. 2A). Flow cytometricanalysis validated cellular internalization of CD86-mCherryby the A20 B-cell lymphoma cells cocultured with CD86-mCherryþ RAW macrophages or DC2.4 dendritic cells (Fig.2B). Because CD28 is not expressed by murine A20 B-celllymphoma, it can be excluded from competing with CTLA4 forB7 molecule engagement and cellular internalization underthe experimental conditions (Fig. 2C). Blocking CTLA4 using aCTLA4 blocking antibody resulted in considerably reduceduptake of sCD86 by human B-cell lymphoma Ly3 and Rajicells, indicating that CTLA4 contributes to CD86 cellularinternalization (Fig. 2D).

Tyrosine 218 in CTLA4 mediates ligand internalization inB cells

To investigate the intracellular tyrosine domain(s) of CTLA4involved in CTLA4-mediated cellular internalization of CD86, wegenerated cell lines stably expressing various human CTLA4constructs, particularly those with mutated tyrosines in the cyto-plasmic tail of CTLA4. Incubating the CTLA4-expressing B cell

lines with human sCD86, we observed that membrane distalY218 in CTLA4 was more critical in the ligand internalizationcompared to the membrane proximal Y201 (Fig. 3). Moreover,mutated CTLA4-Y201F increased sCD86 internalization (Fig. 3).However, CTLA4-Y218F affects ligand internalization in a dom-inant manner because ligand uptake by double-mutation Y201F/Y218 in CTLA4 was comparable with ligand uptake by single-mutation Y218F in CTLA4 (Fig. 3, bottom). These results, takentogether, suggest that CTLA4 expressed on malignant B cellscan interact with and internalize CD86, thereby inhibitingT-cell activation by competing with T-cell costimulatingmolecule CD28.

CD86-CTLA4 activates Tyk2 and STAT3Stimulation of human B-cell lymphoma Ly3 cells with soluble

CD86, a critical factor driving B-cell lymphoma disease progres-sion (21), resulted in immediateCTLA4 tyrosine phosphorylationand STAT3 recruitment by CTLA4 (Fig. 4A). Although the intra-cellular signaling pathways of CTLA4 are not well defined, apotential involvement of the JAK2 tyrosine kinase was indicatedin T cells (22). We showed that sCD86 distinctly stimulatedtyrosine phosphorylation of the JAK family member, Tyk2 (Fig.4B), as well as induced Tyk2 recruitment to form a signalingcomplex with CTLA4 (Fig. 4C). CTLA4 ligation with CD86resulted in STAT3 tyrosine phosphorylation (Fig. 4D), andinduced the DNA-binding activity of STAT3, which is criticallyrequired for target gene transcription (Fig. 4E and F). BecauseSTAT3 is well known for its role in promoting tumor immuno-suppression and inhibiting Th1 antitumor immune responses, weassessed whether stimulation of B-cell lymphoma Ly3 cells withsCD86 would lead to expression of its known downstreamimmune-modulatory genes. Stimulating Ly3 cells with sCD86resulted in induction of STAT3 downstream immunosupressive

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Figure 3.

Tyrosine 218 in CTLA4-mediates ligandinternalization in B cells. Mouse pre-B cells stablyexpressing hCTLA4-GFP constructs, with indicatedtyrosine mutations, were used to assessinternalization of fluorescently labeled humansCD86. Top, schematic structure of hCTLA4 with orwithout mutations at tyrosine phophorylation sites.Red line, mutations site. Bottom, representativeflow cytometry analyses showing internalizationof sCD86 by wild-type and mutated hCTLA4. Theexperiments were repeated three times withsimilar results.






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Figure 4.

CD86-CTLA4 intracellular signaling activates Tyk2 and STAT3 in B-cell lymphoma cells. A, CD86-CTLA4 engagement immediately triggers CTLA4 tyrosinephosphorylation and recruitment of STAT3 in Ly3 cells. Ly3 tumor cells were treated with sCD86, followed by immunoprecipitation with CTLA4 antibody andWestern blotting to detect pTyr-CTLA4 and STAT3. B, Tyk2, but not JAK1, 2, or 3, undergoes tyrosine phosphorylation upon exposure to sCD86. C, Exposureof Ly3 cells to sCD86 results in recruitment of Tyk2 by CTLA4 as assessed by coimmunoprecipitation and Western blotting. D and E, CD86 induces immediateSTAT3 tyrosine phosphorylation as shown by flow cytometry (D) and by EMSA using a radiolabeled dsDNA oligo (SIE) harboring a STAT1 and STAT3 bindingconsensus sequence (E). � , STAT3 supershift with a STAT3-specific antibody. F, RT-PCR shows effects of CTLA4-CD86 engagement on mRNA expression ofSTAT3 target oncogenic genes (left) and immunoregulatory genes (right) in human B-cell lymphoma Ly3 cells, which were stimulated by sCD86 stimulationfor 24 hours. G and H, CTLA4 blockade reduces sCD86-induced STAT3 activation as shown by Western blotting (G) and subsequent effects on STAT3 downstreamgene expression assessed by RT-PCR for mRNA in three B-cell lymphoma cell lines as indicated (H). SD shown. t test: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

Herrmann et al.





CD86-mCherry

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Figure 5.

CD86-CTLA4 engagement promotes B-cell lymphoma proliferation and growth via Tyk2-STAT3. A, CD86 on APCs stimulates lymphoma cell proliferation.CD86-mCherry–expressing RAW macrophages (left) or DC2.4 cells (right) were incubated with CFSEþ A20 lymphoma cells, followed by flow cytometryto assess dividing A20 cells (top). Highly proliferative CFSE-low versus nonproliferative CFSE-high A20 cells were compared for CD86-mCherryinternalization (bottom). B, CTLA4 antibody-blockade significantly reduced A20 lymphoma growth in syngeneic mice. C, CTLA4 blockade in vivo significantlydecreased Ki67þ proliferative activity. Scale for confocal microscopy, 100 mm. Ki67 mean fluorescence quantified. D and E, CTLA4 knockdown in Ly3 B-celllymphoma reduced tumor growth in vivo in a xenograft model (D) and decreased Ki67 expression in tumor tissue analyzed by confocalmicroscopy (E); scale, 50 mm.F, Blocking CTLA4 significantly delayed human B-cell lymphoma growth in immunodeficient mice. G, Blocking CTLA4 in vivo reduced Tyk2 activationand STAT3 recruitment in human lymphoma, as shown by Western blotting using tumor homogenates from the tumors shown in F. H, CTLA4 blockade in humanB-cell lymphoma in vivo inhibits lymphoma oncogenesis, indicated by changes in levels of CD31, Ki67, and cleaved caspase-3þ in the lymphoma tumors. Confocalmicroscopy scale, 100 and 50 mm. CD31, Ki67, and cleaved caspase-3 mean fluorescence quantified. SD shown. t test: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






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Figure 6.

CTLA4-Tyk2-STAT3 oncogenic signaling is active in tumor-associated B cells. A, CTLA4-blockade inhibits tumor growth of B16 melanoma in syngeneicmice. SD shown; t test: � , P < 0.05; �� , P < 0.01. B, Flow cytometric analyses show that CTLA4 antibody blockade inhibits Tyk2 and Stat3 activity as well asexpression of c-Myc oncogene in CD19þ B cells isolated from the TDLNs. C, Reduced c-Myc expression by melanoma-infiltrating CD19þ B cells upon CTLA4blockade was confirmed by confocal microscopy; scale, 20 mm. D, In vivo blockade of CTLA4 induces CD8 T cells melanoma infiltration. E, The tumor-infiltratingCD8 T cells are mostly CD69þ. F, Flow cytometric analyses indicate the effects of CTLA4 blockade on nonmalignant B cells from lymph nodes of A20subcutaneous tumor-bearing mice (n ¼ 4/cohort).


Herrmann et al.




genes, such as IL10 and IL6, as well as inhibition of IFNg expres-sion (Fig. 4F). At the same time, CTLA4 ligation with CD86caused upregulation of STAT3 downstream cancer-promotinggenes in B lymphoma cells, such as BCL2L1 and MMP9, asassessed by RT-PCR (Fig. 4F). Moreover, we were able to dem-onstrate that sCD86-induced STAT3 activation was considerablydecreased upon CTLA4 blockade in human B-cell lymphoma Ly3cells (Fig. 4G). In addition, CTLA4 blockade resulted in signifi-cantly reduced expression of STAT3 target genes tested in varioushuman B-cell lymphoma cell lines (Fig. 4H). Data shown in Fig. 3have identified an unexpected role of CTLA4 in promoting tumorcell survival and proliferation. In addition, CTLA4 intracellularsignaling through Tyk2-STAT3 promotes expression of immuno-suppressive genes while inhibiting the production of Th1 immu-nostimulatory molecules.

CD86-CTLA4 promotes tumor cell growthElevated JAK-STAT3 signaling in tumor cells, including many

types of B lymphomas, has been demonstrated to promote tumorcell proliferation, survival, and resistance to apoptosis (14, 18, 23,24). We therefore assessed whether CTLA4-CD86 ligation wouldincrease B-cell lymphoma tumor cell proliferation. CFSEþ A20lymphoma B cells cocultured with CD86-mCherry–expressingmacrophages or dendritic cells diluted the fluorescent intensityof CFSE dye loaded into lymphoma B cells, indicating inducedlymphoma cell division/proliferation by CD86. Conversely, non-proliferative CFSEhigh lymphoma cells had low CD86-mCherrysignal (Fig. 5A). These findings are indicative of a direct correla-tion between CD86 internalization and mitotic activity of lym-phoma B cells in vitro.

CTLA4 antibody blockade in vivo, employed to inhibitCTLA4 interaction with CD86, significantly reduced tumorgrowth in a syngenic A20 B-cell lymphoma tumor model (Fig.5B). CTLA4 antibody treatment also activated T cells (Supple-mentary Fig. S2). Importantly, Ki67þ proliferative activity wassignificantly reduced in tumors treated with CTLA4 blockingantibodies (Fig. 5C).

Moreover, inhibiting CTLA4 by either silencing CTLA4 inhuman lymphoma tumor cells or treating with CTLA4 blockingantibodies significantly reduced B-cell lymphoma tumor growthin mice lacking T cells and B cells (Fig. 5D–F). Importantly,CTLA4-blockade in human B-cell lymphoma considerablyreduced activation of Janus kinase Tyk2 and recruitment of STAT3by CTLA4 (Fig. 5G), as well as significantly diminished Ki67þ

proliferative activity and increased tumor cell apoptosis, whichwas also associated with disruption of CD31þ tumor vasculature(Fig. 5H). We therefore show that CTLA4 ligation with CD86promotes B-cell lymphoma tumor growth, which is associatedwith Tyk2-STAT3 activation induced by CTLA4. These resultsprovided a molecular mechanism by which CD86 drives B-celllymphoma progression.

CTLA4-STAT3 signaling is active in tumor-associated B cellsA critical role of the tumor-associated B cells in cancer has

been demonstrated in previous pioneering studies (6–11). Theoncogenic effects of tumor-associated B cells are contributed bySTAT3 activity (12, 13). We therefore examined the possibilitythat CTLA4 is expressed by tumor-associated CD19þ B cells andthat signaling via Tyk2-STAT3 is operative in the tumor-asso-ciated B cells, thereby promoting tumor growth. Flow cytome-try analysis of tumor-infiltrating B cells showed that CTLA4 was

expressed by the B cells enriched from B16 tumors (Supple-mentary Fig. S3). Treating B16 melanoma tumor-bearing micewith CTLA4 antibodies significantly inhibited tumor growth(Fig. 6A). Expression of pTyk2, pStat3 and c-Myc by tumor-associated CD19þ B cells was decreased upon CTLA4 blockadein vivo as assessed by flow cytometry (Fig. 6B). The decrease inc-Myc expression in B16 melanoma infiltrating CD19þ B cellsupon administration of CTLA4 blocking antibody was con-firmed by confocal microscopy (Fig. 6C). Furthermore, CTLA4blockade improved the infiltration of activated CD8þCD69þ Tcells into tumor tissue and induced the downregulation ofCD62L by CD3þ T cells in the tumor environment (Fig. 6Dand E; Supplementary Fig. S2).

Moreover, CTLA4 blockade treatment resulted in activation ofCD19þB cells (nonmalignant) in tumor-draining lymphnodes intheA20 subcutaneous tumor-bearingmice (Fig. 6F, top).Notably,the tumor-promoting CD5þCD19þ B-cell population (13) wasconsiderably decreased upon CTLA4 blockade in vivo (Fig. 6F,bottom). Our results with B16 melanoma and A20 lymphomashow that in addition to suppressing T-cell activation, CTLA4signaling also negatively impacts tumor-associated B-cell antitu-mor activity.

DiscussionAlthough our studies focused on the role of CTLA4 in B cells

in cancer, they shed light on fundamental functions of CTLA4in B cells. By internalizing CD86 expressed on antigen-present-ing cells, CTLA4 in B cells can downmodulate T-cell Th1immune responses. Our study has identified a novel cell-intrin-sic pathway by CTLA4 to suppress Th1 immunity throughSTAT3. During normal physiology, inhibition of Th1 immunityis a prerequisite of wound healing, which involves cell prolif-eration, resistance to apoptosis, and angiogenesis. The process-es of wound healing are the same as those in cancer. STAT3 isknown to regulate wound healing and its persistent activationis critical for oncogenesis. Our results reveal that CTLA4 notonly is critical for downmodulating immune responses but alsopromotes cell proliferation, survival, and angiogenesis. STAT3activation in tumor-associated immune cells, including B cellspromotes production of growth factors and other mediators toenhance tumor cell growth (12–14).

We show that upon engagement with CD86, CTLA4 recruitsand activatesTyk2, which is reminiscent of the interactionbetween a cytokine receptor and JAK. Through both geneticsilencing and antibody blockade, our work suggests that CTLA4is a target in B-cell lymphoma tumor cells and in tumor-associated B cells for cancer therapy. However, the potency ofthe antitumor effects by anti-CTLA4 antibody therapy, com-pared with CTLA4 gene silencing, in the B-cell lymphomaxenograft tumor model in the absence of T cells and B cellsis not dramatic. This could be due to the fact that CTLA4 is alsoexpressed in the cell cytoplasm (5) in addition to cell surfaceexpression. Our results further suggest that CTLA4 blockade inconjunction with STAT3 inhibition should increase CTLA4immunotherapy, and CTLA4 blockade treatment for B-celllymphoma has the added advantage of directly inhibitingtumor cell growth/resistance to apoptosis.

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






DisclaimerThe content is solely the responsibility of the authors and does not neces-

sarily represent the official views of the NIH.

Authors' ContributionsConception and design:A.Herrmann,H. Lee, K. Jenkins, T. Blankenstein, H. YuDevelopment of methodology: A. HerrmannAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Herrmann, C. Lahtz, J.Y. Song, W.C. Chan, C. Yue,T. Look, R. M€ulfarth, W. Li, J. Williams, L.E. BuddeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Herrmann, T. Nagao, W.C. Chan, C. Yue, W. LiWriting, review, and/or revision of the manuscript: A. Herrmann, T. Nagao,J.Y. Song, L.E. Budde, S. Forman, L. Kwak, H. YuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Herrmann, J.Y. SongStudy supervision: A. Herrmann, H. Yu

AcknowledgmentsWe thank the dedication of staff members at the flow-cytometry core and

light microscopy core at the Beckman Research Institute at City of HopeComprehensive Cancer Center for their technical assistance. We alsoacknowledge the contribution of staff members at the animal facilities atCity of Hope.

Grant SupportThis work was supported by R01CA122976, R01CA146092,

P50CA107399, the Tim Nesvig Lymphoma Society, V Foundation Transla-tional Research Grant, and by the National Cancer Institute of the NIHunder grant number P30CA033572.

Received March 14, 2016; revised May 4, 2017; accepted July 7, 2017;published OnlineFirst July 17, 2017.

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Herrmann et al.




2017;77:5118-5128. Published OnlineFirst July 17, 2017.Cancer Res Andreas Herrmann, Christoph Lahtz, Toshikage Nagao, et al.

Dependent B-cell Oncogenicity−CTLA4 Promotes Tyk2-STAT3

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