CTLA4 Promotes Tyk2-STAT3–Dependent B-Cell Oncogenicity Andreas Herrmann1, Christoph Lahtz1, Toshikage Nagao1,2, Joo Y

Published OnlineFirst July 17, 2017; DOI: 10.1158/0008-5472.CAN-16-0342 Cancer Microenvironment and Immunology Research

CTLA4 Promotes Tyk2-STAT3–Dependent B-cell Oncogenicity Andreas Herrmann1, Christoph Lahtz1, Toshikage Nagao1,2, Joo Y. Song3, Wing C. Chan3, Heehyoung Lee1, Chanyu Yue1, Thomas Look1, Ronja Mulfarth€ 1, 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-established lymphoma cell proliferation and tumor growth, whereas silencing immune checkpoint for antitumor immune responses. The pro- or antibody-blockade of CTLA4 in B-cell lymphoma tumor cells in tumorigenic function of CTLA4 is believed to be limited to T-cell the absence of T cells inhibits tumor growth. This inhibition was inhibition by countering the activity of the T-cell costimulating accompanied by reduction of Tyk2/STAT3 activity, tumor cell receptor CD28. However, as we demonstrate here, there are two proliferation, and induction of tumor cell apoptosis. The CTLA4– additional roles for CTLA4 in cancer, including via CTLA4 over- Tyk2–STAT3 signal pathway was also active in tumor-associated expression in diverse B-cell lymphomas and in melanoma- nonmalignant B cells in mouse models of melanoma and lym- associated B cells. CTLA4-CD86 ligation recruited and activated phoma. Overall, our results show how CTLA4-induced immune the JAK family member Tyk2, resulting in STAT3 activation and suppression occurs primarily via an intrinsic STAT3 pathway and expression of genes critical for cancer immunosuppression that CTLA4 is critical for B-cell lymphoma proliferation and and tumor growth and survival. CTLA4 activation resulted in survival. Cancer Res; 77(18); 1–11. 2017 AACR.

Introduction approaches to optimize the antitumor efﬁcacy of CTLA4-blocking antibodies. CTL–associated antigen 4 (CTLA4) is well recognized as an The mechanism by which CTLA4 dampens T-cell responses has immune checkpoint, and has emerged as a prominent target for been attributed to the fact that CTLA4 shares identical ligands, cancer immunotherapy (1, 2). CTLA4-blocking antibodies, along B7-1 (CD80)/B7.2 (CD86; refs. 3, 4) on antigen-presenting cells, with PD1 and PD-L1–blocking antibodies, are capable of unleash- with T-cell costimulating receptor CD28. However, whether and ing antitumor immune responses with durable cancer regression how CTLA4 may dampen T-cell activation through cell-intrinsic (1, 2). However, despite being one of the most potent anticancer mechanism remains unknown. In addition, although it is con- drugs, CTLA4-blocking antibodies are unable to signiﬁcantly sidered expressed exclusively by T cells, there are some indications prolong the lives of majority of the treated patients, suggesting that CTLA4 is expressed by certain malignant B cells (5). If CTLA4 an urgent need to further understand CTLA4 biology in cancer, is consistently and highly expressed by B cells in the tumor thereby enabling the development of rational combinatory microenvironment, it would suggest that B cells could also dampen T-cell activation by competing with CD28 for engaging B7-1 (CD80)/B7.2 (CD86) on antigen-presenting cells. However, these concepts have not been formerly tested. 1Department of Onco-Immunology, Beckman Research Institute, City of Hope A critical role of tumor-associated B cells in promoting cancer Comprehensive Cancer Center, Duarte, California. 2Department of Hematology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental survival/resistance to therapies as well as immunosuppression has University, Tokyo, Japan. 3Department of Pathology, City of Hope Comprehen- been reported (6–13). Among several mechanisms, STAT3 has sive Cancer Center, Duarte, California. 4Department of Molecular Medicine, been shown to mediate the cancer promoting activities of tumor- Beckman Research Institute, City of Hope Comprehensive Cancer Center, associated B cells (12, 13). STAT3 is persistently activated in 5 Duarte, California. Hematology Institute, City of Hope Comprehensive Cancer diverse cancers, including many B-cell malignancies (14, 15). Center, Duarte, California. 6Max-Delbruck-Center€ for Molecular Medicine, and STAT3 is critical for upregulating the expression of numerous the Institute of Immunology, Charite Campus Buch, Berlin, Germany. genes involved in cancer cell survival/proliferation, and invasion Note: Supplementary data for this article are available at Cancer Research (16). A standout feature of STAT3 in cancer is that it also promotes Online (http://cancerres.aacrjournals.org/). expression of an array of immunosuppressive genes while inhi- A. Herrmann, C. Lahtz, and T. Nagao contributed equally to this article. biting many Th1 immunostimulatory genes necessary for, induc- Corresponding Authors: Andreas Herrmann, Beckman Research Institute—City ing antitumor T-cell immunity (16–18). STAT3 activity in malig- of Hope, Beckman Building, 1500 East Duarte Road, Duarte, CA 91010-3000. nant B cells has been shown to inhibit the antigen presentation Phone: 626-256-4374, ext. 64428; Fax: 626-256-8708; E-mail: ability of these cells (19). STAT3 is persistently activated in diverse [email protected]; and Hua Yu, [email protected] immune subsets in the tumor microenvironment, including mye- doi: 10.1158/0008-5472.CAN-16-0342 loid cells, B cells, as well as T-cell, inducing immunosuppression 2017 American Association for Cancer Research. and promoting tumor growth (4, 12–15, 20). Nevertheless, the

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upstream molecules/receptors that activate STAT3 in malignant B Table 1. Human diffuse large B-cell lymphoma/NHL tumor samples (IRB14225) cells and in tumor-associated "normal" B and T cells remain to be Diffuse large B-cell lymphoma, DLBCL further explored. In this study, we investigated the potential role Sample Diagnosis Site Age Sex 1 DLBCL Lymph node 72 M of CTLA4 in B cells in promoting tumor progression. Our studies fi 2 DLBCL Lymph node 60 M identi ed a cell-intrinsic immunosuppressive pathway for CTLA4 3 DLBCL Lymph node 77 M and an unexpected function of CTLA4 in promoting tumor cell 4 DLBCL Lymph node 74 M growth and survival. 5 DLBCL Soft tissue 51 M 6 DLBCL Lymph node 58 F Materials and Methods 7 DLBCL GI 39 F 8 DLBCL Lymph node 71 M Mice and cell culture 9 DLBCL Lymph node 32 F For subcutaneous tumor challenge, C57BL/6, Balb/c (The 10 DLBCL Lymph node 19 M Jackson Laboratory) or athymic nude mice (NCI Frederick), were 11 DLBCL Lymph node 72 F injected with 105 B16 melanoma or 2.5 105 A20 lymphoma, NOTE: The human tumor samples included in this study were evaluated by physicians at Department of Pathology of City of Hope. respectively. Athymic nu/nu mice (NCI Frederick) were engrafted with 2 106 Ly3 human lymphoma cells subcutaneously into the flank. After tumors reached 5 to 7 mm in diameter, treatment with taining RPMI1640 medium containing 10% FBS, 10 ng/mL IL3 or 250 mg/dose/mouse CTLA4 blocking antibody (BioXCell) was 10% conditioned medium of WEHI-3B cell line. Mouse WEHI-3B locally administered every other day. cells were grown in Iscove's MDM supplemented with 5% to 10% Human B-cell lymphoma Ly3, U266 cells (kindly provided in 5 FBS, 2 mmol/L L-glutamine, and 2.5 10 mol/L mercaptoetha- 2010 by Dr. Ana Scuto, Beckman Research Institute at the Com- nol. Human CTLA-GFP constructs were introduced by electropo- prehensive Cancer Center at the City of Hope, Duarte, CA), Daudi, ration. Briefly, 3.5 106 BA/F3 cells were resuspended in 800 mL JeKo-1, SU-DHL-6, Raji and RPMI6666 cells (ATCC obtained in cell culture media containing 28 mg vector. Cells were pulsed with 2016) were cultured in IMDM or RPMI medium (Gibco), respec- 200 V for 70 msec and subcultured. tively, human multiple myeloma MM.1S (kindly provided in Human B-cell lymphoma Ly3 cells with knocked down 2016 by Dr. Stephen Forman, Comprehensive Cancer Center at human CTLA4 expression were generated using lentiviral shRNA the City of Hope) and H929 (ATCC) were cultured in DMEM particles obtained from Santa Cruz Biotechnology. Cellular medium supplemented with 10% FBS (Sigma) and 0.05 mol/L introduction of shRNA was carried out according to the manu- mercaptoethanol. Mouse DC2.4 dendritic cells (kindly provided facturer's instructions. in 2008 by Dr. Marcin Kortylewski, Beckman Research Institute at the Comprehensive Cancer Center at the City of Hope), A20 B cell Plasmids lymphoma (ATCC obtained in 2009), and mouse B16 melanoma Plasmid coding for mouse CD86-mCherry was obtained from (kindly provided in 2007 by Dr. Drew Pardoll, The Sidney (GeneCopoeia). Plasmid encoding human CTLA4-GFP was pur- Kimmel Comprehensive Cancer Center at Johns Hopkins School chased from OriGene (RG210150). Site directed mutagenesis was of Medicine, Baltimore, MD) were grown in RPMI1640 (Gibco) performed using QuickChange (Stratagene), resulting in hCTLA4 containing 10% FBS. Mouse RAW264.7 macrophages (ATCC, 0 constructs hCTLA4-Y201F (5 -ctcttacaacaggggtctttgtgaaaatgccccca- obtained in 2010) were cultured in DMEM supplemented with 30;50-tgggggcattttcacaaagacccctgttgtaagag-30) and Y218F (50- 10% FBS. Cells used in this study were routinely freshly thawed, 0 0 gcaatttcagcctttttttattcccatcaatacgcgtacg-3 ;5-cgtacgcgtattgatgggaa- subcultured for up to 3 weeks for desired in vitro studies or in vivo taaaaaaaggctgaaattgc-30). engraftment, tested for mycoplasma contamination and authen- ticated by RT-PCR and flow cytometry. Cell subculture was Generation of soluble human CD86 immediately amplified for long-term storage in liquid nitrogen. Human CD86 gene was obtained from DNASU plasmid repos- itory (clone: HsCD00039473). Soluble human CD86-Fc gene in Study approval pVL1393 vector was transfected into Sf9 cells with BestBac 2.0 Mouse care and experimental procedures with mice were per- Baculovirus Cotransfection kit (Expression Systems). High titer formed under pathogen-free conditions in accordance with established institutional guidance and approved Institutional Animal Care and Use Committee protocols from the Research Animal Table 2. Human follicular lymphoma/NHL tumor samples (IRB14225) Care Committees of the City of Hope. Follicular lymphoma, FL Sample Diagnosis Site Age Sex Patient tumor specimens 1 FL1-2 Lymph node 58 F 2 FL3A Lymph node 62 M This study was performed in accordance with the Helsinki 3 FL3A Lymph node 76 M principles and approved by the Institutional Review Board at 4 FL1-2 Lymph node 72 F City of Hope Medical Center (IRB14225). Informed written 5 FL1-2 Lymph node 71 M consent was obtained. The human tumor samples were evaluated 6 FL3A Lymph node 61 M by physicians at Department of Pathology of City of Hope. 7 FL3A Lymph node 66 F Detailed information is summarized in Tables 1 and 2. 8 FL3A Lymph node 61 F 9 FL3A Lymph node 39 M 10 FL1-2 Lymph node 65 M Generating stable cell lines 11 FL3A Lymph node 55 M To generate BA/F3 cell lines stably expressing human CTLA4 NOTE: The human tumor samples included in this study were evaluated by constructs, murine pro-B-cell line BA/F3 was grown in IL3 con- physicians at Department of Pathology of City of Hope.

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virus was generated and used to infect Tni cells at an MOI of 3 for Electrophoretic mobility shift assay protein production. Cells were harvested 48 hours postinfection, Nuclear extracts from cells were isolated using buffer A contain- centrifuged at 4,000 rpm for 25 minutes, and the filtered super- ing 10 mmol/L HEPES/KOH pH 7.9, 1.5 mmol/L MgCl2, natant was applied to a Protein A resin (GenScript). After PBS 10 mmol/L KCl and buffer C containing 20 mmol/L HEPES/KOH wash, protein was eluted with 0.1 mol/L glycine, pH 3.0 and pH 7.9, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L immediately pH adjusted with 1 mol/L Tris-HCl pH 8.0. Con- EDTA, 25% glycerol; per 2 mL buffer, protease inhibitors at centrated eluate was applied to HiLoad 26/60 Superdex 200 0.2 mmol/L PMSF, 0.5 mmol/L DTT, and 1 mmol/L Na3VO4 column (GE Healthcare) in PBS. Peak fractions were concentrated, were added fresh before use. Cells were washed with PBS, resus- flash frozen, and stored at 80 C. Purity was monitored by pended in buffer A, incubated on ice for 20 minutes and sedi- SDS-PAGE. mented by centrifugation for 20 seconds at 13.2 rpm in a table-top Generated and purified human sCD86 was fluorescently centrifuge. Pellet was resuspended in buffer C, incubated for labeled. Briefly, peptide diluted in 200-mL PBS was activated with 30 minutes on ice and sedimented by centrifugation for 10 min- 0 a 1:10 dilution of 1 mol/L NaHCO3 (20 mL), mixed with a grain of utes at 13.2 rpm. Double-stranded DNA SIE oligo (5 -AGCTT- NHS coupled AlexaFluor 647 (Invitrogen) dissolved in 2 mL CATTTCCCGTAAATCCCTA-30/30AGTAAAGGGCATTTAGGGAT- DMSO (Sigma), and incubated light protected at room temper- TCGA-50 containing STAT1and STAT3 consensus binding site was ature for 1 hour up to 1.5 hours. Gel filtration column was packed radiolabeled with 32P-ATP/32P-CTP using Klenow enzyme (Pro- with G75 Sephadex (GE Healthcare) and fluorescently labeled mega). Nuclear extracts were resuspended at 10 mg with loading sCD86 peptide was eluted by centrifugation for 5 minutes at buffer (50 mmol/L HEPES pH 7.8, 5 mmol/L EDTA pH 8, 1,100 g. 25 mmol/L MgCl2 adjusted to pH 7.8 with 3 mol/L KOH) containing radiolabeled SIE-oligo and separated by PAGE elec- Imaging trophoresis; dried gel was exposed on X-ray film to assess STAT3 Indirect immunoflourescence and IHC were carried out as DNA binding. For supershift analysis, aSTAT3 antibody (C-20X, described previously (19) staining CD3, CD20 (BioLegend), Santa Cruz Biotechnology) was added to nuclear extract at CTLA4, c-Myc, pSTAT3 (Santa Cruz Biotechnology), Hoechst 1 mL/20 mL and incubated on ice for 15 minutes before loading 33342 (Sigma), Ki67 (Vector), CD19, CD31 (BioLegend, BD onto PAGE for electrophoretic separation. Biosciences), pTyk2 and cleaved caspase-3 (Cell Signaling Tech- nology). CFSE was purchased from Invitrogen and CFSE loading PCR into cells was carried out according to the manufacturer's instruc- Transcript amplification was determined from total RNA puri- tions. Imaging was carried out on a confocal microscope Zeiss fied using the RNeasy Kit (Qiagen). cDNA was synthesized using LSM510 Meta. the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed in triplicates using the Chromo4 Real-Time Detector Flow cytometry (Bio-Rad). The human GAPDH housekeeping gene was used as an Cell suspensions isolated from tissue were prepared as internal control to normalize target gene mRNA levels. Primers described previously (20) and stained with different combina- were obtained from SA Biosciences (human BCL2L1: tions of fluorophore-coupled antibodies to CD3, CD4, CD8, PPH00082B-200, human MMP9: PPH00152E-200) or custom- ized from Integrated DNA Technologies IDT (human IL6: hIL6 F: CD19, CD28, CD62L, CD69, CD80, CD86, B220, CTLA4, phos- 0 0 0 pho-Tyr705-Stat3, FoxP3, IFNg, IL4 (BD Biosciences). Antibodies 5 -GTACATCCTCGACGGCATC-3 ,R:5-CCTCTTTGCTGCTTT- CACAC-30, human IL10: hIL10 F: 50-TGCCTAACATGCTTCGA- against c-Myc and pTyk2 were purchased from Cell Signaling 0 0 0 Technology; staining was performed using a fluorescently labeled GATC-3 ,R:5-GTTGTCCAGCTGATCCTTCA-3 , human IFNg: hINFG F: 50-GAGATGACTTCGAAAAGCTGAC-30,R:50-CACTTG- secondary antibody (Invitrogen). Fluorescence data were collect- 0 ed on Accuri or Fortessa flow cytometers (BD Biosciences) and GATGAGTTCATGT ATTGC-3 ). analyzed using FlowJo software (Tree Star). Statistical analysis Statistical analyses were performed using Prism (GraphPad) Immunoblotting, immunoprecipitation software. The overall significance for each graph was calculated Whole cell lysates were prepared using RIPA lysis buffer using the two-tailed Student t test. P values of less than 0.05 were containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, considered statistically significant. 1 mmol/L EDTA, 0.5% NP-40, 1 mmol/L NaF, 15% glycerol, and 20 mmol/L b-glycerophosphate. A protease inhibitor cocktail was added fresh to the lysis buffer (Mini Protease Inhibitor Results Cocktail, Roche). Normalized protein amounts were subjected Malignant B cells express functional CTLA4 to electrophoretic separation by SDS-PAGE, transferred onto To date, CTLA4 regulatory functions are considered only in T nitrocellulose for Western blotting, and subsequently immu- cells (2). However, it has been suggested that CTLA4 is also nodetection was performed using antibodies against STAT3, expressed in certain malignant B cells (5). We therefore assessed Tyk2, PY99 (Santa Cruz Biotechnology), anti-pTyr (clone CTLA4 expression in patient B-cell lymphoma biopsies. We 4G10, Millipore) and b-actin (Sigma). For coimmunoprecipi- observed considerably elevated CTLA4 expression by tumor infil- þ þ tation, CTLA4, JAK1, JAK2, JAK3, Tyk2 antibodies (Santa Cruz trating CD3 T cells as well as in CD20 cells in human B-cell Biotechnology) were used to label rProtein G agarose beads lymphoma tissues (Fig. 1A, top). Compared with normal lymph (Invitrogen), subsequently incubated for 16 hours with whole- node, expression of CTLA4 is significantly increased in lymph cell lysates, subjected to electrophoretic protein separation and node with B-cell lymphoma (Fig. 1A, bottom). We also assessed Western blot detection. CTLA4 expression in two main types of human NHL lymphomas,

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A CD3 CTLA4 Merge CD20 CTLA4 Merge

Normal LN B cell lymphoma 1,200 *** 1,000 CTLA4 800 Hoechst 600 400

MFI:: CTLA4 200 0

B CD20 CTLA4 Hoechst Merge 100

80 DLBCL CTLA4¯ 60 CTLA4+ n = 11 ea. 40

FL [%] CTLA4 frequency 0

C D none 5 min 15 min 60 min 120 min BLANK 0.1 22.7 41.6 70.3 95.9 2nd Antibody CTLA4 SSC-A % of Max. % of

CTLA4 FL4-A::sCD86AF647

E none 2h sCD86

Figure 1. CTLA4 expression and function by B-cell lymphoma cells. A, IHC staining followed by confocal microscopy analyses showing CTLA4 expression in 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 normal human 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 of CTLA4 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-cell lymphoma 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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A A20CFSE RAWCD86mCherry Hoechst Merge

CFSE CD86-mCherry Hoechst

B A20CFSE alone Coculture A20 RAW264.7 & 0.22%A20 21.0% 61.6%

A20 Alone A20 from coculture DC2.4 & A20 6.81% 65.1% CD86-mCherry

FL1-A:: CFSE CD86-mCherry

C BLANK BLANK Isotype Isotype CD80 CD28 % of Max. % of % of Max. % of CD86 CTLA-4

FL1-A:: FITC FL2-A:: PE

D None ctrl. sCD86 sCD86+IgG sCD86+αCTLA4

0.26 22.5 25.8 9.7 SSC-A

FL1-A:: human sCD86FAM 0.14 48.1 50.1 30.6 FL1-A:: sCD86

FL4-A:: ctrl FL4-A:: ctrl FL4-A:: IgG ctrl FL4-A:: CTLA4

Figure 2. CTLA4 contributes to CD86 cellular internalization. A, CTLA4-positive A20 B-cell lymphoma cells uptake CD86 from APCs. CD86-mCherry–expressing RAW macrophages were cocultured with CFSEþ A20 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 dendritic cells (bottom) by CFSEþ A20 cells. C, Flow cytometric analyses of CD80, CD86, CD28, and CTLA4 in murine A20 B-cell lymphoma cells. D, CTLA4 blockade reduces sCD86 internalization by human B-cell lymphoma Ly3 (top) and CTLA4þ Raji (bottom) cells assessed by ﬂow cytometry.

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hCTLA4GFP

Y201 Y201F Y201 Y201F Y218 Y218 Y218F Y218F

- GFP Figure 3. WT Y201F Y218F FF Tyrosine 218 in CTLA4-mediates ligand internalization in B cells. Mouse pre-B cells stably expressing hCTLA4-GFP constructs, with indicated WT Y201F Y218F FF hCTLA4GFP tyrosine mutations, were used to assess internalization of ﬂuorescently labeled human 0.1 0.2 0.3 0.3 sCD86. Top, schematic structure of hCTLA4 with or without mutations at tyrosine phophorylation sites. none Red line, mutations site. Bottom, representative ﬂow cytometry analyses showing internalization of sCD86 by wild-type and mutated hCTLA4. The experiments were repeated three times with similar results. 36.4 52.0 19.4 20.6 shCD86647 FL4-A: AlexaFluor647

FL1-A: GFP

diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma lines with human sCD86, we observed that membrane distal (FL; Tables 1 and 2). We show that CTLA4 is detectable in both Y218 in CTLA4 was more critical in the ligand internalization types of NHL lymphomas (DLBCL, 81% and FL, 36%; Fig. 1B). compared to the membrane proximal Y201 (Fig. 3). Moreover, CTLA4 is also expressed in tested cell lines derived from mutated CTLA4-Y201F increased sCD86 internalization (Fig. 3). human B malignancies, including Ly3 (DLBCL; Fig. 1C; Sup- However, CTLA4-Y218F affects ligand internalization in a dom- plementary Fig. S1A and S1B) and human multiple myeloma inant manner because ligand uptake by double-mutation Y201F/ þ cell lines (Supplementary Fig. S1). CTLA4 B-cell lymphoma Y218 in CTLA4 was comparable with ligand uptake by single- cells rapidly engaged with soluble CD86 (sCD86; Fig. 1D; mutation Y218F in CTLA4 (Fig. 3, bottom). These results, taken Supplementary Fig. S1C), allowing CD86 cellular internaliza- together, suggest that CTLA4 expressed on malignant B cells tion (Fig. 1E). Incubating murine RAW macrophages expres- can interact with and internalize CD86, thereby inhibiting sing ﬂuorescently labeled full-length CD86-mCherry with T-cell activation by competing with T-cell costimulating mouse B-cell lymphoma A20 cells loaded with CFSE resulted molecule CD28. þ in a CD86-mCherry A20 B-cell lymphoma population, as shown by confocal microscopy (Fig. 2A). Flow cytometric CD86-CTLA4 activates Tyk2 and STAT3 analysis validated cellular internalization of CD86-mCherry Stimulation of human B-cell lymphoma Ly3 cells with soluble by the A20 B-cell lymphoma cells cocultured with CD86- CD86, a critical factor driving B-cell lymphoma disease progres- þ mCherry RAW macrophages or DC2.4 dendritic cells (Fig. sion (21), resulted in immediate CTLA4 tyrosine phosphorylation 2B). Because CD28 is not expressed by murine A20 B-cell and STAT3 recruitment by CTLA4 (Fig. 4A). Although the intra- lymphoma, it can be excluded from competing with CTLA4 for cellular signaling pathways of CTLA4 are not well deﬁned, a B7 molecule engagement and cellular internalization under potential involvement of the JAK2 tyrosine kinase was indicated the experimental conditions (Fig. 2C). Blocking CTLA4 using a in T cells (22). We showed that sCD86 distinctly stimulated CTLA4 blocking antibody resulted in considerably reduced tyrosine phosphorylation of the JAK family member, Tyk2 (Fig. uptake of sCD86 by human B-cell lymphoma Ly3 and Raji 4B), as well as induced Tyk2 recruitment to form a signaling cells, indicating that CTLA4 contributes to CD86 cellular complex with CTLA4 (Fig. 4C). CTLA4 ligation with CD86 internalization (Fig. 2D). resulted in STAT3 tyrosine phosphorylation (Fig. 4D), and induced the DNA-binding activity of STAT3, which is critically Tyrosine 218 in CTLA4 mediates ligand internalization in required for target gene transcription (Fig. 4E and F). Because B cells STAT3 is well known for its role in promoting tumor immuno- To investigate the intracellular tyrosine domain(s) of CTLA4 suppression and inhibiting Th1 antitumor immune responses, we involved in CTLA4-mediated cellular internalization of CD86, we assessed whether stimulation of B-cell lymphoma Ly3 cells with generated cell lines stably expressing various human CTLA4 sCD86 would lead to expression of its known downstream constructs, particularly those with mutated tyrosines in the cyto- immune-modulatory genes. Stimulating Ly3 cells with sCD86 plasmic tail of CTLA4. Incubating the CTLA4-expressing B cell resulted in induction of STAT3 downstream immunosupressive

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CTLA4 in B Cells Promotes Oncogenicity via Tyk2-STAT3

A IP: CTLA4 B IP: JAK1 JAK2 JAK3 Tyk2 – + sCD86, 15 min. – + – + – + – + sCD86, 15 min. - pTyr (34 kDa) - pTyr (130 kDa)

- STAT3 (89 kDa) -IgGhc

-IgGhc

C IP: CTLA4 D E +* +– sCD86 – + sCD86, 15 min. - Stat3/3 - Stat1/3 - Tyk2 (130 kDa) - Stat1/1

-IgGhc Max. % of

pY-STAT3 BLANK Isotype Untreated 30 min., 100 μg/mL sCD86 free probe F 2.5 *** 3.5 ** 120 ** 7 *** 1.2 *** 3.0 6 2.0 100 1.0 2.5 5 80 0.8 1.5 2.0 4 mRNA mRNA mRNA mRNA 60 0.6 1.0 1.5 3 γ

IL6 40 0.4 IL10 1.0 2 IFN MMP9

BCL2L1 mRNA 0.5 0.5 20 1 0.2 0 0 0 0 0

G H Raji Daudi Ly3 Raji

140 *** 2.5 ** 4.5 * 3.5 ** 120 4 3 2 3.5 100 2.5 3 1.5 - pY-STAT3 80 2.5 2 mRNA 60 1 2 γ 1.5 - STAT3 1.5 40 IFN 1 BCL2L1 mRNA BCL2L1 mRNA 0.5 BCL2L1 mRNA 1 - β-Actin 20 0.5 0.5 0 0 0 0

Figure 4. CD86-CTLA4 intracellular signaling activates Tyk2 and STAT3 in B-cell lymphoma cells. A, CD86-CTLA4 engagement immediately triggers CTLA4 tyrosine phosphorylation and recruitment of STAT3 in Ly3 cells. Ly3 tumor cells were treated with sCD86, followed by immunoprecipitation with CTLA4 antibody and Western blotting to detect pTyr-CTLA4 and STAT3. B, Tyk2, but not JAK1, 2, or 3, undergoes tyrosine phosphorylation upon exposure to sCD86. C, Exposure of Ly3 cells to sCD86 results in recruitment of Tyk2 by CTLA4 as assessed by coimmunoprecipitation and Western blotting. D and E, CD86 induces immediate STAT3 tyrosine phosphorylation as shown by ﬂow cytometry (D) and by EMSA using a radiolabeled dsDNA oligo (SIE) harboring a STAT1 and STAT3 binding consensus sequence (E). , STAT3 supershift with a STAT3-speciﬁc antibody. F, RT-PCR shows effects of CTLA4-CD86 engagement on mRNA expression of STAT3 target oncogenic genes (left) and immunoregulatory genes (right) in human B-cell lymphoma Ly3 cells, which were stimulated by sCD86 stimulation for 24 hours. G and H, CTLA4 blockade reduces sCD86-induced STAT3 activation as shown by Western blotting (G) and subsequent effects on STAT3 downstream gene 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.

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A B 500 65.1% 92.0% Vehicle A20 start IgG control ) A20 cultured alone 3 400 Anti-CTLA4 A20 from Coculture

300

FL1-A:: CFSE 200 Count

Tumor volume (mm 100 ** ** *** *** A20 CFSE-high *** A20 CFSE-low 0 864 10 2018161412 Days

CD86-mCherry

*** C 2,000 Vehicle IgG control Anti-CTLA4 *** 1,600 1,200 Vehicle Ki67 IgG control Hoechst 800 Anti-CTLA4 MFI:: Ki67 400 0 α D E nt-RNA H IgG CTLA4 800 nt-shRNA, n = 5 )

3 CTLA4-shRNA, n = 5 700 CD31 600 Hoechst 500 400 CTLA4shRNA 300 200 Ki67 Tumor volume (mm Tumor volume ** 100 Hoechst ** ** ** 0 4 86 1816141210 Ki67 Time (days) Hoechst F G 700 Human B cell lymphoma Ly3, cl.casp.3

) local 3 600 Hoechst IgG control 500 IP: pTyr- αCTLA4 * 400 Tyk2 IgG- hc 300 *** 500 ** 500 *** 300 * 250 400 400 IP: - STAT3 200 200 * 300 300 150 Vessel (mm) Tumor volume (mm Tumor volume 100 CTLA4 IgG- hc 200 200

+ 100 0 50 100 MFI:: Ki67 100 864 1210 1614 0 0 0 CD31 MFI:: cl. caspase 3 MFI:: cl. caspase Time (days) IgG αCTLA4

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 cytometry to assess dividing A20 cells (top). Highly proliferative CFSE-low versus nonproliferative CFSE-high A20 cells were compared for CD86-mCherry internalization (bottom). B, CTLA4 antibody-blockade significantly reduced A20 lymphoma growth in syngeneic mice. C, CTLA4 blockade in vivo significantly decreased Ki67þ proliferative activity. Scale for confocal microscopy, 100 mm. Ki67 mean fluorescence quantified. D and E, CTLA4 knockdown in Ly3 B-cell lymphoma reduced tumor growth in vivo in a xenograft model (D) and decreased Ki67 expression in tumor tissue analyzed by confocal microscopy (E); scale, 50 mm. F, Blocking CTLA4 significantly delayed human B-cell lymphoma growth in immunodeficient mice. G, Blocking CTLA4 in vivo reduced Tyk2 activation and STAT3 recruitment in human lymphoma, as shown by Western blotting using tumor homogenates from the tumors shown in F. H, CTLA4 blockade in human B-cell lymphoma in vivo inhibits lymphoma oncogenesis, indicated by changes in levels of CD31, Ki67, and cleaved caspase-3þ in the lymphoma tumors. Confocal microscopy 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.

OF8 Cancer Res; 77(18) September 15, 2017 Cancer Research

CTLA4 in B Cells Promotes Oncogenicity via Tyk2-STAT3

A 1,000 B pTyk2 Vehicle IgG control ) 3 800 Anti-CTLA4

600 pStat3 Isotype/2nd ctrl. Vehicle IgG control

400 Count Anti-CTLA4

Tumor volume (mm Tumor volume 200 * c-Myc ** * * 0 810121614 1820 Days Vehicle IgG ctrl. αCTLA4 C D

Vehicle

CD8 Hoechst c-Myc IgG control CD19 Hoechst

F None IgG αCTLA4 Anti-CTLA4 TDLN 6.07 5.4 15.9

E CD8 CD69 Merge

CD69 LN 5.06 4.23 6.52 Vehicle

TDLN 21.8 17.9 13.4 IgG ctrl.

CD5 LN 3.33 4.7 5.56 αCTLA4

CD8 CD69 CD19 Hoechst

Figure 6. CTLA4-Tyk2-STAT3 oncogenic signaling is active in tumor-associated B cells. A, CTLA4-blockade inhibits tumor growth of B16 melanoma in syngeneic mice. 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 as expression of c-Myc oncogene in CD19þ B cells isolated from the TDLNs. C, Reduced c-Myc expression by melanoma-infiltrating CD19þ B cells upon CTLA4 blockade was confirmed by confocal microscopy; scale, 20 mm. D, In vivo blockade of CTLA4 induces CD8 T cells melanoma infiltration. E, The tumor-infiltrating CD8 T cells are mostly CD69þ. F, Flow cytometric analyses indicate the effects of CTLA4 blockade on nonmalignant B cells from lymph nodes of A20 subcutaneous tumor-bearing mice (n ¼ 4/cohort).

www.aacrjournals.org Cancer Res; 77(18) September 15, 2017 OF9

Herrmann et al.

genes, such as IL10 and IL6, as well as inhibition of IFNg expres- expressed by the B cells enriched from B16 tumors (Supple- sion (Fig. 4F). At the same time, CTLA4 ligation with CD86 mentary Fig. S3). Treating B16 melanoma tumor-bearing mice caused upregulation of STAT3 downstream cancer-promoting with CTLA4 antibodies significantly inhibited tumor growth genes in B lymphoma cells, such as BCL2L1 and MMP9, as (Fig. 6A). Expression of pTyk2, pStat3 and c-Myc by tumor- þ assessed by RT-PCR (Fig. 4F). Moreover, we were able to dem- associated CD19 B cells was decreased upon CTLA4 blockade onstrate that sCD86-induced STAT3 activation was considerably in vivo as assessed by flow cytometry (Fig. 6B). The decrease in þ decreased upon CTLA4 blockade in human B-cell lymphoma Ly3 c-Myc expression in B16 melanoma infiltrating CD19 B cells cells (Fig. 4G). In addition, CTLA4 blockade resulted in signifi- upon administration of CTLA4 blocking antibody was con- cantly reduced expression of STAT3 target genes tested in various firmed by confocal microscopy (Fig. 6C). Furthermore, CTLA4 þ þ human B-cell lymphoma cell lines (Fig. 4H). Data shown in Fig. 3 blockade improved the infiltration of activated CD8 CD69 T have identified an unexpected role of CTLA4 in promoting tumor cells into tumor tissue and induced the downregulation of þ cell survival and proliferation. In addition, CTLA4 intracellular CD62L by CD3 T cells in the tumor environment (Fig. 6D signaling through Tyk2-STAT3 promotes expression of immuno- and E; Supplementary Fig. S2). suppressive genes while inhibiting the production of Th1 immu- Moreover, CTLA4 blockade treatment resulted in activation of þ nostimulatory molecules. CD19 B cells (nonmalignant) in tumor-draining lymph nodes in the A20 subcutaneous tumor-bearing mice (Fig. 6F, top). Notably, þ þ CD86-CTLA4 promotes tumor cell growth the tumor-promoting CD5 CD19 B-cell population (13) was Elevated JAK-STAT3 signaling in tumor cells, including many considerably decreased upon CTLA4 blockade in vivo (Fig. 6F, types of B lymphomas, has been demonstrated to promote tumor bottom). Our results with B16 melanoma and A20 lymphoma cell proliferation, survival, and resistance to apoptosis (14, 18, 23, show that in addition to suppressing T-cell activation, CTLA4 24). We therefore assessed whether CTLA4-CD86 ligation would signaling also negatively impacts tumor-associated B-cell antitu- þ increase B-cell lymphoma tumor cell proliferation. CFSE A20 mor activity. lymphoma B cells cocultured with CD86-mCherry–expressing macrophages or dendritic cells diluted the fluorescent intensity Discussion of CFSE dye loaded into lymphoma B cells, indicating induced Although our studies focused on the role of CTLA4 in B cells lymphoma cell division/proliferation by CD86. Conversely, non- in cancer, they shed light on fundamental functions of CTLA4 proliferative CFSEhigh lymphoma cells had low CD86-mCherry in B cells. By internalizing CD86 expressed on antigen-present- signal (Fig. 5A). These findings are indicative of a direct correla- ing cells, CTLA4 in B cells can downmodulate T-cell Th1 tion between CD86 internalization and mitotic activity of lym- immune responses. Our study has identified a novel cell-intrin- phoma B cells in vitro. sic pathway by CTLA4 to suppress Th1 immunity through CTLA4 antibody blockade in vivo,employedtoinhibit STAT3. During normal physiology, inhibition of Th1 immunity CTLA4 interaction with CD86, significantly reduced tumor is a prerequisite of wound healing, which involves cell prolif- growth in a syngenic A20 B-cell lymphoma tumor model (Fig. eration, resistance to apoptosis, and angiogenesis. The process- 5B). CTLA4 antibody treatment also activated T cells (Supple- þ es of wound healing are the same as those in cancer. STAT3 is mentaryFig.S2).Importantly,Ki67 proliferative activity was known to regulate wound healing and its persistent activation significantly reduced in tumors treated with CTLA4 blocking is critical for oncogenesis. Our results reveal that CTLA4 not antibodies (Fig. 5C). only is critical for downmodulating immune responses but also Moreover, inhibiting CTLA4 by either silencing CTLA4 in promotes cell proliferation, survival, and angiogenesis. STAT3 human lymphoma tumor cells or treating with CTLA4 blocking activation in tumor-associated immune cells, including B cells antibodies significantly reduced B-cell lymphoma tumor growth promotes production of growth factors and other mediators to in mice lacking T cells and B cells (Fig. 5D–F). Importantly, enhance tumor cell growth (12–14). CTLA4-blockade in human B-cell lymphoma considerably We show that upon engagement with CD86, CTLA4 recruits reduced activation of Janus kinase Tyk2 and recruitment of STAT3 þ and activatesTyk2, which is reminiscentoftheinteraction by CTLA4 (Fig. 5G), as well as significantly diminished Ki67 between a cytokine receptor and JAK. Through both genetic proliferative activity and increased tumor cell apoptosis, which þ silencing and antibody blockade, our work suggests that CTLA4 was also associated with disruption of CD31 tumor vasculature is a target in B-cell lymphoma tumor cells and in tumor- (Fig. 5H). We therefore show that CTLA4 ligation with CD86 associated B cells for cancer therapy. However, the potency of promotes B-cell lymphoma tumor growth, which is associated the antitumor effects by anti-CTLA4 antibody therapy, com- with Tyk2-STAT3 activation induced by CTLA4. These results pared with CTLA4 gene silencing, in the B-cell lymphoma provided a molecular mechanism by which CD86 drives B-cell xenograft tumor model in the absence of T cells and B cells lymphoma progression. is not dramatic. This could be due to the fact that CTLA4 is also expressed in the cell cytoplasm (5) in addition to cell surface CTLA4-STAT3 signaling is active in tumor-associated B cells expression. Our results further suggest that CTLA4 blockade in A critical role of the tumor-associated B cells in cancer has conjunction with STAT3 inhibition should increase CTLA4 – been demonstrated in previous pioneering studies (6 11). The immunotherapy, and CTLA4 blockade treatment for B-cell oncogenic effects of tumor-associated B cells are contributed by lymphoma has the added advantage of directly inhibiting STAT3 activity (12, 13). We therefore examined the possibility þ tumor cell growth/resistance to apoptosis. that CTLA4 is expressed by tumor-associated CD19 Bcellsand that signaling via Tyk2-STAT3 is operative in the tumor-associated B cells, thereby promoting tumor growth. Flow cytome- Disclosure of Potential Conflicts of Interest try analysis of tumor-infiltrating B cells showed that CTLA4 was No potential conflicts of interest were disclosed.

OF10 Cancer Res; 77(18) September 15, 2017 Cancer Research

CTLA4 in B Cells Promotes Oncogenicity via Tyk2-STAT3

Disclaimer Acknowledgments The content is solely the responsibility of the authors and does not neces- We thank the dedication of staff members at the ﬂow-cytometry core and sarily represent the ofﬁcial views of the NIH. light microscopy core at the Beckman Research Institute at City of Hope Comprehensive Cancer Center for their technical assistance. We also Authors' Contributions acknowledge the contribution of staff members at the animal facilities at Conception and design: A. Herrmann, H. Lee, K. Jenkins, T. Blankenstein, H. Yu City of Hope. Development of methodology: A. Herrmann Acquisition 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. Mulfarth,€ W. Li, J. Williams, L.E. Budde Grant Support Analysis and interpretation of data (e.g., statistical analysis, biostatistics, This work was supported by R01CA122976, R01CA146092, computational analysis): A. Herrmann, T. Nagao, W.C. Chan, C. Yue, W. Li P50CA107399, the Tim Nesvig Lymphoma Society, V Foundation Transla- Writing, review, and/or revision of the manuscript: A. Herrmann, T. Nagao, tional Research Grant, and by the National Cancer Institute of the NIH J.Y. Song, L.E. Budde, S. Forman, L. Kwak, H. Yu under grant number P30CA033572. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Herrmann, J.Y. Song Received March 14, 2016; revised May 4, 2017; accepted July 7, 2017; Study supervision: A. Herrmann, H. Yu published OnlineFirst July 17, 2017.

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www.aacrjournals.org Cancer Res; 77(18) September 15, 2017 OF11

CTLA4 Promotes Tyk2-STAT3−Dependent B-cell Oncogenicity

Andreas Herrmann, Christoph Lahtz, Toshikage Nagao, et al.

Cancer Res Published OnlineFirst July 17, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-0342

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