CTLA4 promotes€Tyk2-STAT3 dependent B cell oncogenecity...checkpoint, and has emerged as a...

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Running title: CTLA4 in B cells promotes oncogenicity via Tyk2-STAT3

Title: CTLA4 promotes Tyk2-STAT3 dependent B cell oncogenicity

Andreas Herrmann1*†, Christoph Lahtz1†, Toshikage Nagao1,5†, Joo Y. Song2, Wing C. Chan2,

Heehyoung Lee1, Chanyu Yue1, Thomas Look1, Ronja Mülfarth1, Wenzhao Li1, Kurt Jenkins3,

John Williams3, Lihua E. Budde4, Stephen Forman4, Larry Kwak4, Thomas Blankenstein6 and

Hua Yu1*

1Department of Onco-Immunology, Beckman Research Institute at City of Hope Comprehensive

Cancer Center, Duarte, CA 91010, USA.

2Department of Pathology, City of Hope Comprehensive Cancer Center.

3Department of Molecular Medicine, Beckman Research Institute at City of Hope

Comprehensive Cancer Center, Duarte, CA 91010, USA.

4Hematology Institute, City of Hope Comprehensive Cancer Center.

5Department of Hematology, Graduate School of Medical and Dental Science, Tokyo Medical

and Dental University, Tokyo, Japan.

6Max-Delbrück-Center for Molecular Medicine, and the Institute of Immunology, Charité Campus

Buch, Berlin, Germany

† These authors contribute equally to this work.

Key words: STAT3, CTLA4, Tyk2, B cell lymphoma

*Corresponding authors: Andreas Herrmann or Hua Yu, Department of Immuno-Oncology,

Beckman Research Institute at City of Hope Comprehensive Cancer Center, Duarte, CA 91010,

USA. Phone: 1.626.256.4673; Fax: 1.626.256.8708; E-mail: [email protected] ;

[email protected]

Financial support: This work was supported by R01CA122976, R01CA146092,

P50CA107399, the Tim Nesvig Lymphoma Society, V Foundation Translational Research

Grant, and by the National Cancer Institute of the National Institutes of Health under grant

number P30CA033572.

on April 15, 2020. © 2017 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 17, 2017; DOI: 10.1158/0008-5472.CAN-16-0342

mailto:[email protected]


http://cancerres.aacrjournals.org/

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Abstract

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is a well-established immune checkpoint

for antitumor immune responses. The pro-tumorigenic function of CTLA4 is believed to be

limited to T cell inhibition by countering the activity of the T cell co-stimulating receptor CD28.

However, as we demonstrate here, there are two additional roles for CTLA4 in cancer, including

via CTLA4 overexpression in diverse B cell lymphomas and in melanoma-associated B cells.

CTLA4-CD86 ligation recruited and activated the JAK family member Tyk2, resulting in STAT3

activation and expression of genes critical for cancer immunosuppression and tumor growth and

survival. CTLA4 activation resulted in lymphoma cell proliferation and tumor growth, whereas

silencing or antibody-blockade of CTLA4 in B cell lymphoma tumor cells in the absence of T

cells inhibits tumor growth. This inhibition was accompanied by reduction of Tyk2/STAT3

activity, tumor cell proliferation, and induction of tumor cell apoptosis. The CTLA4-Tyk2-STAT3

signal pathway was also active in tumor-associated non-malignant B cells in mouse models of

melanoma and lymphoma. Overall, our results show how CTLA4 induced immune suppression

occurs primarily via an intrinsic STAT3 pathway and that CTLA4 is critical for B cell lymphoma

proliferation and survival.




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Introduction

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is well recognized as an immune

checkpoint, and has emerged as a prominent target for cancer immunotherapy (1, 2). CTLA4

blocking antibodies, along with PD1 and PD-L1 blocking antibodies, are capable of unleashing

anti-tumor immune responses with durable cancer regression (1, 2). However, despite being

one of the most potent anticancer drugs, CTLA4 blocking antibodies are unable to significantly

prolong the lives of majority of the treated patients, suggesting an urgent need to further

understand CTLA4 biology in cancer, thereby enabling the development of rational combinatory

approaches to optimize the antitumor efficacy of CTLA4 blocking antibodies.

The mechanism by which CTLA4 dampens T cell responses has been attributed to the fact that

CTLA4 shares identical ligands, B7-1 (CD80)/B7.2 (CD86) (3, 4) on antigen-presenting cells,

with T cell co-stimulating receptor CD28. However, whether and how CTLA4 may dampen T

cell activation through cell-intrinsic mechanism remains unknown. In addition, although it is

considered expressed exclusively by T cells, there are some indications that CTLA4 is

expressed by certain malignant B cells (20). If CTLA4 is consistently and highly expressed by B

cells in the tumor 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.

A critical role of tumor-associated B cells in promoting cancer survival/resistance to therapies as

well as immunosuppression has been reported (5-12). Among several mechanisms, STAT3 has

been shown to mediate the cancer promoting activities of tumor-associated B cells (11, 12).

STAT3 is persistently activated in diverse cancers, including many B cell malignancies (13, 14).

STAT3 is critical for upregulating the expression of numerous genes involved in cancer cell

survival/proliferation, and invasion (15). A standout feature of STAT3 in cancer is that it also




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promotes expression of an array of immunosuppressive genes while inhibiting many Th1

immunostimulatory genes necessary for inducing antitumor T cell immunity (15-17). STAT3

activity in malignant B cells has been shown to inhibit the antigen presentation ability of these

cells (18). STAT3 is persistently activated in diverse immune subsets in the tumor

microenvironment, including myeloid cells, B cells, as well as T cell, inducing

immunosuppression and promoting tumor growth (4, 11-14, 19). Nevertheless, the upstream

molecules/receptors that activate STAT3 in malignant B cells and in tumor-associated “normal”

B and T cells remain to be further explored. In this study we investigated the potential role of

CTLA4 in B cells in promoting tumor progression. Our studies identified a cell-intrinsic

immunosuppressive pathway for CTLA4 and an unexpected function of CTLA4 in promoting

tumor cell growth and survival.




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Materials and methods

Mice and cell culture

For subcutaneous tumor challenge, C57BL/6, Balb/c (The Jackson Laboratory) or athymic nude

mice (NCI Frederick), were injected with 105 B16 melanoma or 2.5x105 A20 lymphoma,

respectively. Athymic nu/nu mice (NCI Frederick) were engrafted with 2x106 Ly3 human

lymphoma cells s.c. into the flank. After tumors reached 5-7 mm in diameter, treatment with 250

g/dose/mouse CTLA4 blocking antibody (BioXCell) was locally administered every other day.

Human B cell lymphoma Ly3, U266 cells (kindly provided in 2010 by Dr. Ana Scuto, Beckman

Research Institute at the Comprehensive Cancer Center at the City of Hope, CA), Daudi, JeKo-

1, SU-DHL-6, Raji and RPMI6666 cells (ATCC obtained in 2016) were cultured in IMDM or

RPMI medium (Gibco), respectively, human multiple myeloma MM.1S (kindly provided in 2016

by Dr. Stephen Forman, Comprehensive Cancer Center at the City of Hope, CA) and H929

(ATCC) were cultured in DMEM medium supplemented with 10% FBS (Sigma) and 0.05 M

mercaptoethanol. Mouse DC2.4 dendritic cells (kindly provided in 2008 by Dr. Marcin

Kortylewski, Beckman Research Institute at the Comprehensive Cancer Center at the City of

Hope, CA), A20 B cell lymphoma (ATCC obtained in 2009), and mouse B16 melanoma (kindly

provided in 2007 by Dr. Drew Pardoll, The Sidney Kimmel Comprehensive Cancer Center at

Johns Hopkins School of Medicine, Baltimore, MD) were grown in RPMI1640 (Gibco) containing

10% FBS. Mouse RAW264.7 macrophages (ATCC obtained in 2010) were cultured in DMEM

supplemented with 10% FBS. Cells used in this study were routinely freshly thawed,

subcultured for up to three weeks for desired in vitro studies or in vivo engraftment, tested for

mycoplasma contamination and authenticated by RT-PCR and flow cytometry. Cell subculture

was immediately amplified for long term storage in liquid nitrogen.

Study approval




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Mouse care and experimental procedures with mice were performed under pathogen-free

conditions in accordance with established institutional guidance and approved IACUC protocols

from the Research Animal Care Committees of the City of Hope.

Patient tumor specimens

This study was performed in accordance with the Helsinki principles and approved by the

institutional review board at City of Hope Medical Center (IRB14225). Informed written consent

was obtained. The human tumor samples were evaluated by physicians at Department of

Pathology of City of Hope. Detailed information is summarized in tables 1 and 2 (Tables T1, T2).

Generating stable cell lines

To generate BA/F3 cell lines stably expressing human CTLA4 constructs, murine pro-B cell line

BA/F3 was grown in IL-3 containing RPMI 1640 medium containing 10% FBS, 10 ng/ml IL-3 or

10% conditioned medium of WEHI-3B cell line. Mouse WEHI-3B cells were grown in Iscove's

MDM supplemented with 5-10% FBS, 2 mM L-glutamine, and 2.5 x 10-5 M mercaptoethanol.

Human CTLA-GFP constructs were introduced by electroporation. Briefly, 3.5x106 BA/F3 cells

were resuspended in 800 l cell culture media containing 28 g vector. Cells were pulsed with

200 V for 70 msec and subcultured.

Human B cell lymphoma Ly3 cells with knocked down human CTLA4 expression were

generated using lentiviral shRNA particles obtained from Santa Cruz. Cellular introduction of

shRNA was carried out according to the manufacturer’s instructions.

Plasmids

Plasmid coding for mouse CD86-mCherry was obtained from (GeneCopoeia). Plasmid encoding

human CTLA4-GFP was purchased from OriGene (RG210150). Site directed mutagenesis was

performed using QuickChange (Stratagene) resulting in hCTLA4 constructs hCTLA4-Y201F




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(5’-ctcttacaacaggggtctttgtgaaaatgccccca-3’; 5’-tgggggcattttcacaaagacccctgttgtaagag-3’) and

Y218F (5’-gcaatttcagcctttttttattcccatcaatacgcgtacg-3’; 5’-

cgtacgcgtattgatgggaataaaaaaaggctgaaattgc-3’).

Generation of soluble human CD86

Human CD86 gene was obtained from DNASU plasmid repository (clone: HsCD00039473).

Soluble human CD86-Fc gene in pVL1393 vector was transfected into Sf9 cells with BestBac

2.0 Baculovirus Cotransfection kit (Expression Systems, Davis, CA). High titer virus was

generated and used to infect Tni cells at an MOI of 3 for protein production. Cells were

harvested 48 h post-infection, centrifuged at 4,000 rpm for 25 min, and the filtered supernatant

was applied to a Protein A resin (GenScript). After PBS wash, protein was eluted with 0.1 M

glycine, pH 3.0 and immediately pH adjusted with 1 M Tris-HCl pH 8.0. Concentrated eluate

was applied to HiLoad 26/60 Superdex 200 column (GE Healthcare) in PBS. Peak fractions

were concentrated, flash frozen, and stored at -80º C. Purity was monitored by SDS-PAGE.

Generated and purified human sCD86 was fluorescently labeled. Briefly, peptide diluted in 200

l PBS was activated with a 1:10 dilution of 1 M NaHCO3 (20 l), mixed with a grain of NHS

coupled AlexaFluor 647 (Invitrogen) dissolved in 2 l DMSO (Sigma), and incubated light

protected at room temperature for 1 h up to 1.5 h. Gel filtration column was packed with G75

Sephadex (GE Healthcare) and fluorescently labeled sCD86 peptide was eluted by

centrifugation for 5 min at 1,100 xg.

Imaging

Indirect immunoflourescence and immunohistochemistry were carried out as described

previously (18) staining CD3, CD20 (BioLegend), CTLA4, c-Myc, pSTAT3 (Santa Cruz),

Hoechst33342 (Sigma), Ki67 (Vector), CD19, CD31 (BioLegend, BD Biosciences), pTyk2 and

cleaved caspase 3 (Cell Signalling Technologies). CFSE was purchased from Invitrogen and




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CFSE loading into cells was carried out according to the manufacturer’s instructions. Imaging

was carried out on a confocal microscope Zeiss LSM510 Meta.

Flow cytometry

Cell suspensions isolated from tissue were prepared as described previously (19) and stained

with different combinations of fluorophore-coupled antibodies to CD3, CD4, CD8, CD19, CD28,

CD62L, CD69, CD80, CD86, B220, CTLA4, phospho-Tyr705-Stat3, FoxP3, IFN, IL-4 (BD

Biosciences). Antibodies against c-Myc and pTyk2 were purchased from Cell Signalling

Technologies; staining was performed using a fluorescently labeled secondary antibody

(Invitrogen). Fluorescence data were collected on Accuri or Fortessa flow cytometers (BD) and

analyzed using FlowJo software (Tree Star).

Immunoblotting, immunoprecipitation

Whole cell lysates were prepared using RIPA lysis buffer containing 50 mM Tris (pH 7.4), 150

mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM NaF, 15% glycerol, and 20 mM -glycerophosphate.

A protease inhibitor cocktail was added fresh to the lysis buffer (Mini Protease Inhibitor Cocktail,

Roche). Normalized protein amounts were subjected to electrophoretic separation by SDS-

PAGE, transferred onto nitrocellulose for Western blotting, and subsequently immunodetection

was performed using antibodies against STAT3, Tyk2, PY99 (Santa Cruz), anti-pTyr (clone

4G10, Millipore) and -actin (Sigma). For co-immunoprecipitation, CTLA4, JAK1, JAK2, JAK3,

Tyk2 antibodies (Santa Cruz) were used to label rProtein G agarose beads (Invitrogen),

subsequently incubated for 16 h with whole cell lysates, subjected to electrophoretic protein

separation and Western blot detection.

Electrophoretic mobility shift assay (EMSA)




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Nuclear extracts from cells were isolated using buffer A containing 10 mM HEPES/KOH pH7.9,

1.5 mM MgCl2, 10 mM KCl and buffer C containing 20 mM HEPES/KOH pH 7.9, 420 mM NaCl,

1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol; per 2 ml buffer, protease inhibitors at 0.2 mM

PMSF, 0.5 mM DTT and 1 mM Na3VO4 were added fresh prior to use. Cells were washed with

PBS, resuspended in buffer A, incubated on ice for 20 min and sedimented by centrifugation for

20 sec at 13.2 rpm in a table-top centrifuge. Pellet was resuspended in buffer C, incubated for

30 min on ice and sedimented by centrifugation for 10 min at 13.2 rpm. Double-stranded DNA

SIE oligo (5’-AGCTTCATTTCCCGTAAATCCCTA-3’/3’AGTAAAGGGCATTTAGGGATTCGA-5’

containing STAT1and STAT3 consensus binding site was radiolabeled with 32P-ATP/32P-CTP

using Klenow enzyme (Promega). Nuclear extracts were resuspended at 10 g with loading

buffer (50 mM HEPES pH7.8, 5 mM EDTA pH8, 25 mM MgCl2 adjusted to pH 7.8 with 3 M

KOH) containing radiolabeled SIE-oligo and separated by PAGE electrophoresis; dried gel was

exposed on x-ray film to assess STAT3 DNA binding. For supershift analysis, STAT3 antibody

(C-20X, Santa Cruz) was added to nuclear extract at 1 l/20 l and incubated on ice for 15 min

prior to loading onto PAGE for electrophoretic separation.

Polymerase Chain Reaction

Transcript amplification was determined from total RNA purified using RNeasy Kit (QIAGEN).

cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was

performed in triplicates using the Chromo4 Real-Time Detector (Bio-Rad). The human GAPDH

housekeeping gene was used as an internal control to normalize target gene mRNA levels.

Primers were obtained from SA Biosciences (human BCL2L1: PPH00082B-200, human MMP9:

PPH00152E-200) or customized from Integrated DNA Technologies IDT (human IL-6: hIL-6 F:

5’-GTACATCCTCGACGGCATC-3’, R: 5’-CCTCTTTGCTGCTTTCACAC-3’, human IL-10: hIL-

10 F: 5’-TGCCTAACATGCTTCGAGATC-3’, R: 5’-GTTGTCCAGCTGATCCTTCA-3’, human




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IFN: hINFG F: 5’-GAGATGACTTCGAAAAGCTGAC-3’, R: 5’-CACTTGGATGAGTTCATGT

ATTGC-3’).

Statistics

Statistical analyses were performed using Prism (Graph-Pad) software. The overall significance

for each graph was calculated using two-tailed student’s t-test. P values of less than 0.05 were

considered statistically significant.




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Results

Malignant B cells express functional CTLA4

To date, CTLA4 regulatory functions are considered only in T cells (2). However, it has been

suggested that CTLA4 is also expressed in certain malignant B cells (20). We therefore

assessed CTLA4 expression in patient B cell lymphoma biopsies. We observed considerably

elevated CTLA4 expression by tumor infiltrating CD3+ T cells as well as in CD20+ cells in human

B cell lymphoma tissues (Fig. 1A, upper panels). Compared to normal lymph node, expression

of CTLA4 is significantly increased in lymph node with B cell lymphoma (Fig. 1A, lower panels).

We also assessed CTLA4 expression in two main types of human NHL lymphomas, DLBCL and

follicular lymphoma (Tables T1, T2). We show that CTLA4 is detectable in both types of NHL

lymphomas (DLBCL, 81% and FL, 36%) (Fig. 1B).

CTLA4 is also expressed in tested cell lines derived from human B malignancies, including Ly3

(DLBCL) (Fig. 1C, Supplementary Fig. S1A, B) and human multiple myeloma cell lines

(Supplementary Fig. S1). CTLA4+ B cell lymphoma cells rapidly engaged with soluble CD86

(sCD86) (Fig. 1D, Supplementary Fig. S1C), allowing CD86 cellular internalization (Fig. 1E).

Incubating murine RAW macrophages expressing fluorescently labeled full-length CD86-

mCherry with mouse B cell lymphoma A20 cells loaded with CFSE resulted in a CD86-mCherry+

A20 B cell lymphoma population, as shown by confocal microscopy (Fig. 2A). Flowcytometric

analysis validated cellular internalization of CD86-mCherry+ by the A20 B cell lymphoma cells

co-cultured with CD86-mCherry+ RAW macrophages or DC2.4 dendritic cells (Fig. 2B). Since

CD28 is not expressed by murine A20 B cell lymphoma, it can be excluded from competing with

CTLA4 for B7 molecule engagement and cellular internalization under the experimental

conditions (Fig. 2C). Blocking CTLA4 employing a CTLA4 blocking antibody resulted in




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considerably reduced uptake of sCD86 by human B cell lymphoma Ly3 and Raji cells, indicating

that CTLA4 contributes to CD86 cellular internalization (Fig. 2D).

Tyrosine 218 in CTLA4-mediates ligand internalization in B cells

To investigate the intracellular tyrosine domain(s) of CTLA4 involved in CTLA4-mediated

cellular internalization of CD86, we generated cell lines stably expressing various human CTLA4

constructs, particularly those with mutated tyrosines in the cytoplasmic tail of CTLA4. Incubating

the CTLA4-expressing B cell lines with human sCD86, we observed that membrane distal Y218

in CTLA4 was more critical in the ligand internalization compared to the membrane proximal

Y201 (Fig. 3). Moreover, mutated CTLA4-Y201F increased sCD86 internalization (Fig. 3).

However, CTLA4-Y218F affects ligand internalization in a dominant manner since ligand uptake

by double-mutation Y201F/Y218 in CTLA4 was comparable to ligand uptake by single-mutation

Y218F in CTLA4 (Fig. 3, lower panels). These results, taken together, suggest that CTLA4

expressed on malignant B cells can interact with and internalize CD86, thereby inhibiting T cell

activation by competing with T cell co-stimulating molecule CD28.

CD86-CTLA4 activates Tyk2 and STAT3

Stimulation of human B cell lymphoma Ly3 cells with soluble CD86, a critical factor driving B cell

lymphoma disease progression (21), resulted in immediate CTLA4 tyrosine phosphorylation and

STAT3 recruitment by CTLA4 (Fig. 4A). Although the intracellular signaling pathways of CTLA4

are not well defined, a potential involvement of the JAK2 tyrosine kinase was indicated in T cells

(22). We showed that sCD86 distinctly stimulated tyrosine phosphorylation of the JAK family

member, Tyk2 (Fig. 4B), as well as induced Tyk2 recruitment to form a signaling complex with

CTLA4 (Fig. 4C). CTLA4 ligation with CD86 resulted in STAT3 tyrosine phosphorylation (Fig.

4D), and induced the DNA-binding activity of STAT3, which is critically required for target gene

transcription (Fig. 4E, F). Because STAT3 is well known for its role in promoting tumor




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immunosuppression and inhibiting Th1 antitumor immune responses, we assessed whether

stimulation of B cell lymphoma Ly3 cells with sCD86 would lead to expression of its known

downstream immune-modulatory genes. Stimulating Ly3 cells with sCD86 resulted in induction

of STAT3 downstream immunosupressive genes, such as IL-10 and IL-6, as well as inhibition of

IFN expression (Fig. 4F). At the same time, CTLA4 ligation with CD86 caused upregulation of

STAT3 downstream cancer-promoting genes in B lymphoma cells, such as BclXL and MMP9, as

assessed by RT-PCR (Fig. 4F). Moreover, we were able to demonstrate that sCD86-induced

STAT3 activation was considerably decreased upon CTLA4 blockade in human B cell

lymphoma Ly3 cells (Fig. 4G). In addition, CTLA4 blockade resulted in significantly reduced

expression of STAT3 target genes tested in various human B cell lymphoma cell lines (Fig. 4H).

Data shown in Figure 3 have identified an unexpected role of CTLA4 in promoting tumor cell

survival and proliferation. In addition, CTLA4 intracellular signaling through Tyk2-STAT3

promotes expression of immunosuppressive genes while inhibiting the production of Th1

immunostimulatory molecules.

CD86-CTLA4 promotes tumor cell growth

Elevated JAK-STAT3 signaling in tumor cells, including many types of B lymphomas, has been

demonstrated to promote tumor cell proliferation, survival and resistance to apoptosis (13, 17,

23, 24). We therefore assessed whether CTLA4-CD86 ligation would increase B cell lymphoma

tumor cell proliferation. CFSE+ A20 lymphoma B cells co-cultured with CD86-mCherry

expressing macrophages or dendritic cells diluted the fluorescent intensity of CFSE dye loaded

into lymphoma B cells, indicating induced lymphoma cell division/proliferation by CD86.

Conversely, non-proliferative CFSEhigh lymphoma cells had low CD86-mCherry signal (Fig. 5A).

These findings are indicative of a direct correlation between CD86 internalization and mitotic

activity of lymphoma B cells in vitro.




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CTLA4 antibody blockade in vivo, employed to inhibit CTLA4 interaction with CD86, significantly

reduced tumor growth in a syngenic A20 B cell lymphoma tumor model (Fig. 5B). CTLA4

antibody treatment also activated T cells (Supplementary Figure S2). Importantly, Ki67+

proliferative activity was significantly reduced in tumors treated with CTLA4 blocking antibodies

(Fig. 5C).

Moreover, inhibiting CTLA4 by either silencing CTLA4 in human lymphoma tumor cells or

treating with CTLA4 blocking antibodies significantly reduced B cell lymphoma tumor growth in

mice lacking T cells and B cells (Fig. 5D-F). Importantly, CTLA4-blockade in human B cell

lymphoma considerably reduced activation of Janus kinase Tyk2 and recruitment of STAT3 by

CTLA4 (Fig. 5G), as well as significantly diminished Ki67+ proliferative activity and increased

tumor cell apoptosis, which was also associated with disruption of CD31+ tumor vasculature

(Fig. 5H). We therefore show that CTLA4 ligation with CD86 promotes B cell lymphoma tumor

growth, which is associated with Tyk2-STAT3 activation induced by CTLA4. These results

provided a molecular mechanism by which CD86 drives B cell lymphoma progression.

CTLA4-STAT3 signaling is active in tumor-associated B cells

A critical role of the tumor-associated B cells in cancer has been demonstrated in previous

pioneering studies (5-10). The oncogenic effects of tumor-associated B cells are contributed by

STAT3 activity (11, 12). We therefore examined the possibility that CTLA4 is expressed by

tumor-associated CD19+ B cells and that signaling via Tyk2-STAT3 is operative in the tumor-

associated B cells, thereby promoting tumor growth. Flowcytometry analysis of tumor-infiltrating

B cells showed that CTLA4 was expressed by the B cells enriched from B16 tumors

(Supplementary Fig. S3). Treating B16 melanoma tumor-bearing mice with CTLA4 antibodies

significantly inhibited tumor growth (Fig. 6A). Expression of pTyk2, pStat3 and c-Myc by tumor-

associated CD19+ B cells was decreased upon CTLA4 blockade in vivo as assessed by




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flowcytometry (Fig. 6B). The decrease in c-Myc expression in B16 melanoma infiltrating CD19+

B cells upon administration of CTLA4 blocking antibody was confirmed by confocal microscopy

(Fig. 6C). Furthermore, CTLA4 blockade improved the infiltration of activated CD8+CD69+ T

cells into tumor tissue and induced the downregulation of CD62L by CD3+ T cells in the tumor

environment (Fig. 6D, E, Supplementary Fig. S2).

Moreover, CTLA4 blockade treatment resulted in activation of CD19+ B cells (non-malignant) in

tumor draining lymph nodes in the A20 subcutaneous tumor bearing mice (Fig. 6F, upper

panels). Notably, the tumor-promoting CD5+CD19+ B cell population (12) was considerably

decreased upon CTLA4 blockade in vivo (Fig. 6F, lower panels). Our results with B16

melanoma and A20 lymphoma show that in addition to suppressing T cell activation, CTLA4

signaling also negatively impacts tumor-associated B cell antitumor activity.




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Discussion

Although our studies focused on the role of CTLA4 in B cells in cancer, they shed light on

fundamental functions of CTLA4 in B cells. By internalizing CD86 expressed on antigen

presenting cells, CTLA4 in B cells can downmodulate T cell Th1 immune responses. Our study

has identified a novel cell-intrinsic pathway by CTLA4 to suppress Th1 immunity through

STAT3. During normal physiology, inhibition of Th1 immunity is a prerequisite of wound

healing, which involves cell proliferation, resistance to apoptosis and angiogenesis. The

processes of wound healing are the same as those in cancer. STAT3 is known to regulate

wound healing and its persistent activation is critical for oncogenesis. Our results reveal that

CTLA4 not only is critical for downmodulating immune responses but also promotes cell

proliferation, survival and angiogenesis. STAT3 activation in tumor-associated immune cells

including B cells promotes production of growth factors and other mediators to enhance tumor

cell growth (11-13).

We show that upon engagement with CD86, CTLA4 recruits and activatesTyk2, which is

reminiscent of the interaction between a cytokine receptor and JAK. Through both genetic

silencing and antibody blockade our work suggests that CTLA4 is a target in B cell lymphoma

tumor cells and in tumor-associated B cells for cancer therapy. However, the potency of the

antitumor effects by anti-CTLA4 antibody therapy, compared CTLA4 gene silencing, in the B cell

lymphoma xenograft tumor model in the absence of T cells and B cells is not dramatic. This

could be due to the fact that CTLA4 is also expressed in the cell cytoplasm (20) in addition to

cell surface expression. Our results further suggest that CTLA4 blockade in conjunction with

STAT3 inhibition should increase CTLA4 immunotherapy, and CTLA4 blockade treatment for B

cell lymphoma has the added advantage of directly inhibiting tumor cell growth/resistance to

apoptosis.




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Acknowledgements

We thank the dedication of staff members at the flow cytometry core and light microscopy core

at the Beckman Research Institute at City of Hope Comprehensive Cancer Center for their

technical assistance. We also acknowledge the contribution of staff members at the animal

facilities at City of Hope. The content is solely the responsibility of the authors and does not

necessarily represent the official views of the National Institutes of Health.




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18. Cheng F, Wang H, Horna P, Wang Z, Shah B, Sahakian E, et al. Stat3 inhibition

augments the immunogenicity of B-cell lymphoma cells, leading to effective antitumor

immunity. Cancer Res 2012;72:4440-8.

19. Herrmann A, Priceman SJ, Kujawski M, Xin H, Cherryholmes GA, Zhang W, et al.

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells. J Clin

Invest 2014;124:2977-87.

20. Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of CTLA-4 function. Annu

Rev Immunol 2006;24:65-97.

21. Suvas S, Singh V, Sahdev S, Vohra H, Agrewala JN. Distinct role of CD80 and CD86 in

the regulation of the activation of B cell and B cell lymphoma. J Biol Chem

2002;277:7766-75.

22. Chikuma S, Murakami M, Tanaka K, Uede T. Janus kinase 2 is associated with a box 1-

like motif and phosphorylates a critical tyrosine residue in the cytoplasmic region of

cytotoxic T lymphocyte associated molecule-4. J Cell Biochem 2000;78:241-50.

23. Roschewski M, Staudt LM, Wilson WH. Diffuse large B-cell lymphoma-treatment

approaches in the molecular era. Nat Rev Clin Oncol 2014;11:12-23.

24. Shaffer AL, 3rd, Young RM, Staudt LM. Pathogenesis of human B cell lymphomas. Annu

Rev Immunol 2012;30:565-610.




20

Diffuse large B cell lymphoma, DLBCL

Sample Diagnosis Site Age Sex

1 DLBCL Lymph node 72 M




5 DLBCL Soft tissue 51 M

6 DLBCL Lymph node 58 F

7 DLBCL GI 39 F





Table T1. Human diffuse large B cell lymphoma/NHL tumor samples (IRB14225). The

human tumor samples included in this study were evaluated by physicians at Department of

Pathology of City of Hope.




21

Follicular lymphoma, FL

Sample Diagnosis Site Age Sex

1 FL1-2 Lymph node 58 F

2 FL3A Lymph node 62 M


4 FL1-2 Lymph node 72 F

5 FL1-2 Lymph node 71 M


7 FL3A Lymph node 66 F

8 FL3A Lymph node 61 F


10 FL1-2 Lymph node 65 M


Table T2. Human follicular lymphoma/NHL tumor samples (IRB14225). The human tumor

samples included in this study were evaluated by physicians at Department of Pathology of City

of Hope.




22

Figure 1. CTLA4 expression and function by B cell lymphoma cells. (A)

Immunohistochemical staining followed by confocal microscopy analyses showing CTLA4

expression in CD3+ T cells and CD20+ cells in human B cell lymphoma tissues. Indicated areas

(white boxes) magnified. Scale, 50 m (upper panels). CTLA4 expression in normal human

lymph node vs. lymph node with B cell lymphoma, shown by confocal images and quantification

(lower panels). (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=11 for both tumor types) (right).

Scale, 50 m. (C) CTLA4 surface expression by human B cell lymphoma cell line Ly3 assessed

by flowcytometry. (D) Flowcytometry and (E) confocal microscopy showing cellular

internalization of soluble CD86 by Ly3 cells. Scale, 10 m.




23

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

co-cultured with CFSE+ A20 cells. Cellular internalization of full-length CD86-mCherry by A20

cells was visualized by confocal microscopy. Scale, 10 m. (B) Flowcytometric quantitative

analysis showing CD86-mCherry cellular internalization expressed by RAW macrophages

(upper panels) or dendritic cells (lower panels) by CFSE+ A20 cells. (C) Flowcytometric

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 (upper panels) and

CTLA4+ Raji cells (lower panels) assessed by flowcytometry.




24

Figure 3. Tyrosine 218 in CTLA4-mediates ligand internalization in B cells. Mouse pre-B

cells stably expressing hCTLA4-GFP constructs, with indicated tyrosine mutations, were used to

assess internalization of fluorescently labeled human sCD86. Top, schematic structure of

hCTLA4 with or without mutations at tyrosine phophorylation sites. Red line indicates mutations

site. Bottom, representative flowcytometry analyses showing internalization of sCD86 by wild-

type and mutated hCTLA4. The experiments were repeated three times with similar results.




25

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 co-immunoprecipitation and Western blotting. (D) (E) CD86 induces

immediate STAT3 tyrosine phosphorylation as shown by flowcytometry (D), and by EMSA

employing a radiolabeled dsDNA oligo (SIE) harboring a STAT1 and STAT3 binding consensus

sequence (E). *) indicates 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 panels) and immune regulatory genes (right panels) in human B cell lymphoma Ly3

cells, which were stimulated by sCD86 stimulation for 24 h. (G) CTLA4 blockade reduces

sCD86 induced STAT3 activation as shown by Western blotting and (H) subsequent effects on

STAT3 downstream gene expression assessed by RT-PCR for mRNA in three B cell lymphoma

cell lines as indicated SD shown. T-test: *) P < 0.05, **) P < 0.01; ***) P < 0.001.




26

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 panels) or DC2.4 cells (right panels) were incubated with

CFSE+ A20 lymphoma cells followed by flowcytometry to assess dividing A20 cells (upper

panels). Highly proliferative CFSE-low versus non-proliferative CFSE-high A20 cells were

compared for CD86-mCherry internalization (lower panels). (B) CTLA4 antibody-blockade

significantly reduced A20 lymphoma growth in syngeneic mice. (C) CTLA4 blockade in vivo

significantly decreased Ki67+ proliferative activity. Scale for confocal microscopy, 100 m. Ki67

mean fluorescence quantified. (D) CTLA4 knockdown in Ly3 B cell lymphoma reduced tumor

growth in vivo in a xenograft model and decreased Ki67 expression in tumor tissue analyzed by

confocal microscopy (E). Scale, 50 m. (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 3+ in the lymphoma tumors. Confocal microscopy scale, 100 m and 50

m. CD31, Ki67, and cleaved caspase 3 mean fluorescence quantified. SD shown. T-test: *) P

< 0.05 **) P < 0.01; ***) P < 0.001.




27

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) Flowcytometric analyses show that CTLA4 antibody blockade

inhibits Tyk2 and Stat3 activity as well as expression of c-Myc oncogene in CD19+ B cells

isolated from the TDLNs. (C) Reduced c-Myc expression by melanoma infiltrating CD19+ B

cells upon CTLA4 blockade was confirmed by confocal microscopy. Scale, 20 m. (D) In vivo

blockade of CTLA4 induces CD8 T cells melanoma infiltration. (E) The tumor- infiltrating CD8 T

cells are mostly CD69+. (F) Flowcytometric analyses indicate the effects of CTLA4 blockade on

non-malignant B cells from lymph nodes of A20 subcutaneous tumor bearing mice (n=4/cohort).




A CD3 CTLA4 Merge CD20 CTLA4 Merge

MF

I:: C

TL

A4

0

1,200

1,000

800

600

400

200

***

CTLA4

Hoechst

normal LN B cell lymphoma

Herrmann et al. Fig. 1

B CD20 CTLA4 Hoechst Merge

DLBCL

FL

0

20

40

60

80

100

CT

LA

4 fre

quency [%

]

CTLA4¯

CTLA4+

FL

n=11 ea.

% o

f M

ax.

CTLA4

BLANK

2nd antibody

CTLA4

0.1 22.7 41.6 70.3 95.9

none 5 min 15 min 60 min 120 min

FL4-A::sCD86AF647

SS

C-A

sCD86

Hoechst

DIC

2h sCD86 none

D

E

C





isotype

% o

f M

ax.

FL1-A:: FITC

BLANK

CD86

CD80

isotype

% o

f M

ax.

FL2-A:: PE

BLANK

CTLA-4

CD28

C

61.6% 0.22% 21.0%

RAW264.7 &

A20

6.81%

65.1%

DC2.4 & A20

A20

FL1-A:: CFSE CD86-mCherry

CD

86

-mC

herr

y

A20 from co-culture

A20CFSE alone co-culture

A20 alone

B

D

FL1-A:: human sCD86FAM

none ctrl. sCD86 sCD86+IgG sCD86+aCTLA4

SS

C-A

0.26 22.5 25.8 9.7

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

FL

1-A

:: s

CD

86

30.6 0.14 48.1 50.1

CFSE

CD86-mCherry

Hoechst

A20CFSE RAWCD86mCherry Hoechst Merge A





Y201

Y218

WT

Y201F

Y218

Y201

Y218F

Y201F

Y218F

Y201F Y218F FF

hCTLA4GFP

WT Y201F Y218F FF

FL1-A: GFP

FL

4-A

: A

lexa

Flu

or6

47

none

shCD86647

- GFP

0.1 0.2 0.3 0.3

36.4 52.0 19.4 20.6

hCTLA4GFP




isotype

% o

f M

ax.

pY-STAT3

BLANK

30 min., 100 mg/ml sCD86

untreated

A

- pTyr (34 kDa)

– + sCD86, 15 min.

IP: CTLA4 B

- STAT3 (89 kDa)

- IgGhc

- pTyr (130 kDa)

sCD86, 15 min.

IP:

- IgGhc

– + – + – + – +

Tyk2 JAK1 JAK2 JAK3

C D

- Tyk2 (130 kDa)

– + sCD86, 15 min.

IP: CTLA4

- IgGhc

E + – + sCD86

- Stat3/3 - Stat1/3

- Stat1/1

*

free probe

F

0

0.5

1.0

1.5

3.0

3.5

MM

P9

mR

NA

2.5

2.0

**

0

0.5

1.0

1.5

2.0

2.5

Bcl2

L1 m

RN

A

***


0

0.2

0.4

0.6

1.2

IFNg

mR

NA

1.0

0.8

***

0

20

40

60

120

IL-6

mR

NA

100

80

**

0

1

2

3

7

IL-1

0 m

RN

A

6

4

***

5

G

- pY-STAT3

- STAT3

- b-actin

H

Bcl2

L1 m

RN

A

0

20

40

60

140

100

80

120

***

Raji

Bcl2

L1 m

RN

A

0

0.5

2.5

1.5

1

2

Daudi

Bcl2

L1 m

RN

A

0

1.5

2

2.5

4.5

3.5

3

4

Ly3

**

0.5

1

*

IFNg

mR

NA

0

0.5

1

1.5

3.5

2.5

2

3

**

Raji




CD86-mCherry

65.1% 92.0%

FL1-A:: CFSE

A20 cultured alone A20 start

A20 from co-culture

A20 CFSE-low A20 CFSE-high

100

200

300

400

500

0 4 6 8 10 12

Time [days]

Tu

mo

r vo

lum

e [m

m3]

IgG control

aCTLA4

Human B cell lymphoma Ly3,

local 600

700

14 16

*

*

*

- pTyr IP:

Tyk2 - IgGhc

IP:

CTLA4

- STAT3

- IgGhc

Co

unt

vehicle IgG control anti-CTLA4

Hoechst Ki67

IgG control vehicle

anti-CTLA4

***

***

2,000

1,600

1,200

800

400

0

MF

I:: K

i67

A B

C

D E

100

200

300

500

0 12 14 16 18 20

Tu

mo

r vo

lum

e [m

m3]

400

days

vehicle

IgG control

anti-CTLA4

4 6 8 10

*** *** *** ** **


nt-RNA

CTLA4shRNA

Ki67

Hoechst

0 4

800

700

600

500

400

300

200

100

6 8 10 12 14 16 18

**

Time [days]

Tu

mo

r vo

lum

e [m

m3]

** ** **

nt-shRNA, n=5

CTLA4-shRNA, n=5

F G

IgG aCTLA4

Hoechst CD31

Hoechst Ki67

Hoechst

cl.casp.3

IgG

aCTLA4

0 50

100 150 200 250 300

0

200

400

500

100

300

0

200

400

500

100

300

CD

31

+ v

esse

l [m

m]

MF

I:: K

i67

MF

I:: cl. C

asp

ase 3

*** *** **

H




A B

Co

unt

pTyk2

pStat3

vehicle

IgG control

anti-CTLA4

Isotype/2nd ctrl.

c-Myc 200

400

600

1,000

0 20

Tu

mo

r vo

lum

e [m

m3]

800

days

vehicle

IgG control

anti-CTLA4

8 10 12 18 16 14

*

* ** *


C

vehicle

IgG control

anti-CTLA4

c-Myc

CD19

Hoechst

D vehicle IgG ctrl. aCTLA4

CD8

Hoechst

E

CD8

CD69

Hoechst

vehicle

IgG ctrl.

aCTLA4

CD8 CD69 Merge

CD19

CD

69

none IgG aCTLA4 TDLN

LN

CD

5

TDLN

LN

6.07 5.4 15.9

5.06 4.23 6.52

21.8 17.9 13.4

3.33 4.7 5.56

F




Published OnlineFirst July 17, 2017.Cancer Res Andreas Herrmann, Christoph Lahtz, Toshikage Nagao, et al. CTLA4 promotes Tyk2-STAT3 dependent B cell oncogenecity

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