Yuliya Pylayeva-Gupta , Shipra Das , Jesse S. …...2015/12/28 · Yuliya Pylayeva-Gupta1,3, Shipra Das1, Jesse S. Handler1, Cristina H. Hajdu2, Maryaline Coffre2, Sergei Koralov2,

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IL-35 producing B cells promote the development of pancreatic neoplasia

Yuliya Pylayeva-Gupta1,3, Shipra Das1, Jesse S. Handler1, Cristina H. Hajdu2,

Maryaline Coffre2, Sergei Koralov2, Dafna Bar-Sagi1

1Department of Biochemistry and Molecular Pharmacology; 2Department of

Pathology, New York University School of Medicine, New York, NY, 10016, USA

3Present address: Department of Genetics, Lineberger Comprehensive Cancer

Center, University of North Carolina School of Medicine, Chapel Hill, NC, 27514,

USA.

Correspondence: Dafna Bar-Sagi, Ph.D., Department of Biochemistry and

Molecular Pharmacology, NYU School of Medicine, 550 First Avenue, Smilow 201,

New York, NY 10016; FAX 646-501-6721; email: [email protected]

Keywords: KRas; pancreas; cancer; B cells; Interleukin-10; Interleukin-35

Running title: B cells play a pro-tumorigenic role in pancreatic neoplasia

Conflict of interest: The authors have no conflict of interest to disclose.

Research. on October 15, 2020. © 2015 American Association for Cancercancerdiscovery.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 December 29, 2015; DOI: 10.1158/2159-8290.CD-15-0843

http://cancerdiscovery.aacrjournals.org/

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ABSTRACT

A salient feature of pancreatic ductal adenocarcinoma (PDA) is an abundant

fibroinflammatory response characterized by the recruitment of immune and

mesenchymal cells and the consequent establishment of a pro-tumorigenic

microenvironment. Here we report the prominent presence of B cells in human

pancreatic intraepithelial neoplasia (PanIN) and PDA lesions as well as in

oncogenic K-Ras-driven pancreatic neoplasms in the mouse. The growth of

orthotopic pancreatic neoplasms harboring oncogenic K-Ras was significantly

compromised in B cell-deficient mice (μMT), and this growth deficiency could be

rescued by the reconstitution of a CD1dhighCD5+ B cell subset. The pro-

tumorigenic effect of B cells was mediated by their expression of IL-35 through a

mechanism involving IL-35-mediated stimulation of tumor cell proliferation. Our

results identify a previously unrecognized role for IL-35-producing CD1dhighCD5+

B cells in the pathogenesis of pancreatic cancer and underscore the potential

significance of a B cell/IL-35 axis as a therapeutic target.

SIGNIFICANCE

This study identifies a B cell subpopulation that accumulates in the pancreatic

parenchyma during early neoplasia and is required to support tumor cell

growth. Our findings provide a rationale for exploring B cell-based targeting

approaches for the treatment of pancreatic cancer.




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INTRODUCTION

Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive disease with a

dismal 5- year survival rate of 6% and a poor response to all existing therapies.

The development of PDA is initiated by mutations in the KRas oncogene followed

by inactivating mutations and deletion of tumor suppressor genes including

TRP53, CDKN2A, and SMAD4 (1). The role of these alterations in the initiation

and progression of PDA has been attributed to cell-intrinsic processes that are

critical for malignant transformation, including the bypass of proliferative barriers,

metabolic adaptation and metastatic dissemination.

In addition to these genetically-driven cell intrinsic changes, a key

pathophysiological aspect of PDA is the recruitment of host immune cells into the

tumor microenvironment. Investigations into the functional relevance of discrete

tumor infiltrating immune cell subtypes have uncovered a multitude of

immunomodulatory mechanisms mediated by recruited cells. For example, tumor

associated macrophages and myeloid-derived suppressor cells have been

shown to promote pancreatic tumorigenesis through the suppression of anti-

tumor immunity via expression of heme oxygenase-1 and arginase, respectively

(2-4). CD4+ T cells repress the anti-tumor activity of CD8+ cytotoxic T cells from

the onset of pancreatic neoplasia (5). Likewise, regulatory subset of CD4+ T cells

promotes progression of pancreatic neoplasia by suppressing anti-tumor T cell

immunity in mice immunized with Listeria monocytogenes (6). Furthermore, PDA

associated inflammation potentiates differentiation of immune cell subsets, such

as Th17 T cells and plasmacytoid dendritic cells, that can enhance tumor cell




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growth (7, 8). Significantly, these mechanisms are engaged at very early stages

of disease development and represent attractive targets for therapeutic

intervention.

We have previously shown that the formation of preinvasive lesions known as

pancreatic intraepithelial neoplasia (PanIN) is accompanied by the recruitment of

B cells into the pancreatic parenchyma (3). In the present study we sought to

determine whether this immune cell population plays a role in neoplastic

progression. Our findings identify a B cell subset that contributes to pancreatic

cancer pathogenesis through a paracrine mechanism that promotes the

proliferation of the transformed epithelium.

RESULTS

To investigate the role of B cells in pancreatic tumorigenesis, we first assessed

whether their presence is linked to pancreatic neoplasia in human and mouse.

Prominent B cell infiltrates were detected in proximity to human PanIN lesions as

well as in pancreata of LSL-KrasG12D;p48Cre (KC) mice (Fig. 1A). Furthermore the

implantation of pancreatic ductal epithelial cells expressing oncogenic KRas

(KRasG12D-PDEC) into wild-type (WT) pancreata led to the accumulation of B

cells in regions adjacent to the newly established neoplastic lesions (Fig. 1A)

suggesting an instructive role for the transformed epithelium in B cell recruitment.

We reasoned that the infiltration of neoplastic lesions by B cells would be

mediated by chemotactic cues with the most relevant being the main B cell

chemoattractant CXCL13. Consistent with this postulate, CXCL13 was detected




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in the fibroinflammatory stroma surrounding human and mouse PanIN lesions

(Fig. 1B and C; and Supplementary Fig. S1A and B), and treatment of mice

with anti-CXCL13 blocking antibody resulted in decreased accumulation of B

cells in pancreata of KC mice and mice orthotopically implanted with GFP-

KRasG12D-PDEC (Supplementary Fig. S1C-F). To further characterize the

CXCL13-expressing cell population, qPCR analysis was performed on FACS-

sorted cells from pancreata of KC mice. Using the immune marker CD45 and the

fibroblast marker CD140 (PDGFR), we found that the expression of CXCL13 was

restricted to the fibroblast fraction (CD45-CD140+) of the isolated cells (Fig. 1D).

In agreement with this finding, double immunofluorescent staining revealed that

CXCL13 expressing cells were positive for the mesenchymal marker vimentin

(Fig. 1B and C, insets). Another cell population that could potentially contribute

to CXCL13 production is dendritic cells (9). However, we did not detect CXCL13

mRNA in intra-pancreatic dendritic cells (CD45+CD11c+) (Fig. 1D). Together,

these results indicate that in the context of evolving pancreatic neoplasia, stromal

fibroblasts are induced to secrete CXCL13, thereby promoting the infiltration of B

cells into the pancreatic tumor microenvironment. These observations are

consistent with recent findings documenting that fibroblast-mediated production

of CXCL13 potentiates recruitment of B cells in a prostate cancer model (10).

The physiological relevance of this recruitment event is suggested by the fact

that anti-CXCL13 treatment of mice orthotopically implanted with GFP-KRasG12D-

PDEC resulted in the reduced growth of the orthotopic lesions (Supplementary

Fig. S1G and H).




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To directly analyze the functional significance of B cells in pancreatic

tumorigenesis, GFP-KRasG12D-PDEC were implanted into pancreata of μMT mice

which lack functional B cells or syngeneic WT control animals. Analysis of

pancreata at 2 weeks post-implantation revealed a significant reduction in the

abundance of GFP-KRasG12D-PDEC-derived lesions in μMT mice in comparison

to WT mice (Fig. 1E and F). A similar difference was observed at four weeks

post-implantation (Supplementary Fig. S2A and B). To determine whether the

compromised growth of the neoplastic cells in μMT mice is a direct consequence

of B cell loss, WT B cells were adoptively transferred into μMT animals. Two

days post adoptive transfer, the mice were orthotopically implanted with GFP-

KRasG12D-PDEC and pancreata and spleens were harvested 2 weeks thereafter

(Supplementary Figure S2C). The defect in growth of GFP-KRasG12D-PDEC in

μMT mice was rescued to a significant extent by the adoptive transfer of WT B

cells, and was accompanied by de novo infiltration of transferred B cells (Fig. 1E

and F and Supplementary Fig. S2D), consistent with an essential role for B

cells in establishing a pro-tumorigenic environment. As B lymphocytes were also

observed in the vicinity of neoplastic lesions formed as a consequence of the

concordant pancreatic expression of oncogenic KrasG12D and mutant p53R172H

(Supplementary Fig. S2E), we examined their functional significance in this

setting using cells derived from pdx-1Cre;LSL-KrasG12D;LSL-p53R172H/+ (KPC) mice

(11). Tumors formed by KPC cells that were orthotopically implanted into

pancreata of μMT mice were of significantly reduced size compared to orthotopic

tumors formed in WT pancreata (Supplementary Fig. S2F). These findings




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along with those reported by the accompanying papers (12, 13) suggest that the

presence of B cells might be required to support both early and more advanced

stages of pancreatic tumorigenesis.

Studies conducted in mouse models of squamous carcinomas have

demonstrated that humoral immunity, which is associated with the production of

immunoglobulins by mature B cells, can facilitate tumorigenesis predominantly

through a mechanism involving Fcγ receptor-dependent activation of myeloid

cells (14). To evaluate the role of B cells in myeloid cell activation in the context

of pancreatic tumorigenesis, we analyzed CD45+CD11b+F4/80+ macrophages for

expression of markers specific for either M1 or M2 (tumor-associated

macrophage, TAM) phenotype. We found that, in μMT mice with orthotopic

implants of GFP-KRasG12D-PDEC or GFP-KPC-PDEC, there was a decrease in

the prevalence of TAM-like CD206-expressing intra-pancreatic macrophages and

a corresponding increase in M1-like CD86 positive macrophages

(Supplementary Fig. S3A-D). These observations are consistent with earlier

findings demonstrating that B cell depletion leads to the repolarization of tumor

associated macrophages. To investigate the potential relevance of Fcγ receptor-

dependent activation of macrophages to the observed B cell dependence of

neoplastic growth, we examined the prevalence of antibody-producing plasma

cells in control p48Cre and KC mice. We observed a significant increase in

CD19low/-B220low/-CD138+ plasma cells in the spleens of KC animals (Fig. 2A and

Supplementary Fig. S4A). Concordantly, a significant increase in the proportion

of mature marginal zone B cells (plasma cell precursors) was detected in spleens




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of KC mice as compared to controls (Supplementary Fig. S4B and C),

consistent with an increase in systemic inflammation in mice with pancreatic

cancer (15). However, there was no increase in the abundance of plasma cells in

the pancreatic microenvironment of KC animals (Fig. 2A), suggesting that tumor

infiltrating B cells might modulate pancreatic neoplasia by means other than

immunoglobulin production.

Recent studies addressing the function of B cells in autoimmune disorders

have demonstrated that B cell-mediated cytokine release can alter disease

progression (16). In particular, a subset of cytokine-producing

CD19+CD1dhighCD5+ B cells has been shown to impart immunological tolerance

in autoimmune disease and to promote progression of breast and squamous

carcinomas (17, 18). We found that CD1dhighCD5+ B cells are expanded in

pancreata of KC and orthotopically implanted mice as compared to p48Cre

animals (Fig. 2B and Supplementary Fig. 4SD). To investigate if this B cell

subset contributes to growth of GFP-KRasG12D-PDEC in vivo,

CD19+CD1dhighCD5+ or CD19+CD1dlowCD5- cells were adoptively transferred into

μMT mice (Supplementary Fig. S5A and B). Pancreata were then orthotopically

injected with GFP-KRasG12D-PDEC and harvested for analysis at 2 weeks post-

implantation. While the efficiency of the adoptive transfer was the same for both

B cell subsets, only CD19+CD1dhighCD5+ cells could effectively rescue the

defective growth of GFP-KRasG12D-PDEC in μMT mice (Fig. 2C and D and

Supplementary Fig. S5C). Based on these observations we conclude that




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CD1dhighCD5+ B cells play an essential role in the development of pancreatic

neoplasia.

A critical functional output of CD1dhighCD5+ subtype has been reported to be

the expression of immunosuppressive cytokine IL-10 (19, 20). Consistent with

this attribute, B cell-specific IL-10 expression was detected in both mouse and

human pancreatic cancer (Supplementary Fig. S6A-D). To test whether the

observed B cell-mediated growth promoting effect is IL-10 dependent, WT or

IL10-/- B cells (derived from spleens of WT or IL10-/- mice, respectively) were

adoptively transferred into μMT mice. Two days after B cell transfer, mice were

injected with GFP-KRasG12D-PDEC and cells were allowed to grow for 2 weeks.

The successful transfer of B cells was confirmed using flow cytometry

(Supplementary Fig. S6E). We found that IL10-/- B cells were capable of

rescuing the growth of GFP-KRasG12D-PDEC in vivo to the same extent as WT B

cells (Fig. 2E and F). Thus Il-10 expression is dispensable for the growth

promoting effect of B cells on neoplastic lesions.

It has been recently shown that, in the context of autoimmune and infectious

diseases, CD1dhighCD5+ B cells can confer their immune modulatory effects via

expression of the cytokine IL-35 (a heterodimer, consisting of protein subunits

p35 and EBI3, encoded by genes IL12a and Ebi3, respectively) (21, 22).

Significantly, IL-35 has been found to be upregulated in sera of pancreatic cancer

patients (23). Analysis of KC pancreata revealed that IL12a expression is

primarily confined to B cells and, in particular, to the CD1dhighCD5+ B cell

subpopulation (Fig. 3A and B). A similar pattern of expression was observed for




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Ebi3 transcript (Fig 3C and D). Furthermore, B cell-specific expression of p35

was detected by immunofluorescence in samples of mouse as well as in human

PanIN lesions (Fig. 3E and F and Supplementary Fig. S7A and B). Since the

p35 subunit of IL-35 can combine with p40 (IL12b) and EBI3 can combine with

p28 (IL27) to form IL-12 and IL-27 respectively, we tested the expression of

these subunits in intra-pancreatic B cells. Neither total B cells nor CD1dhighCD5+

subpopulation of B cells isolated from pancreata of KC mice expressed IL12b or

IL27 to an appreciable degree (Supplementary Fig. S7C and D). To directly test

the functional significance of IL-35, WT or IL12a -/- B cells (derived from spleens

of WT or IL12a-/- mice, respectively) were adoptively transferred into μMT mice

(Supplementary Fig. S8), followed by orthotopic implantation of GFP-KRasG12D-

PDEC. As shown in Fig. 3G and H, IL12a -/- B cells failed to rescue the growth

of GFP-KRasG12D-PDEC in vivo suggesting that the B cell-dependent neoplastic

expansion requires IL-35 production. IL-35 has been previously reported to

stimulate the proliferation of pancreatic cancer cell lines (24). We therefore tested

the impact of B cell-mediated IL-35 production on the proliferation of GFP-

KRasG12D-PDEC. As shown in Fig. 3I and J, the absence of B cells was

accompanied by a reduction in epithelial cell proliferation, which was rescued by

WT but not IL12a-/- B cells. No changes in apoptosis were observed under these

conditions as judged by cleaved caspase staining (data not shown). Based on

these observations, we propose that expression of IL-35 by CD1dhighCD5+ is

required for the proliferative expansion of KRasG12D-harboring neoplastic lesions

in vivo.




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DISCUSSION

Understanding the cellular and molecular underpinnings of PDA-associated

immune modulation is a prerequisite for the development of immunotherapy-

based targeting approaches for this deadly malignancy. Our current work

identifies a B cell subset as an important driver of pancreatic tumorigenesis.

Specifically, we demonstrate that, in the context of pancreatic neoplasia, B cells

of CD19+CD1dhighCD5+ cell surface phenotype play a pro-tumorigenic role

through the production of IL-35. In a model of experimental autoimmune

encephalomyelitis (EAE), activation of TLR4 and CD40 has been shown to

induce the upregulation of mRNAs encoding subunits of IL-35 (IL12a and Ebi3)

by B cells(25). By analogy, it is plausible that activation of TLR4 and CD40 could

modulate IL-35 production in B cells in pancreatic cancer, as both TLR4 and

CD40 are upregulated on stromal cells in the pancreatic cancer milieu and

inhibition of TLR4 protects against pancreatic cancer(8). While we have shown

that IL-35 can stimulate the proliferation of tumor cells, one of the IL-35

receptors, gp130(22), is expressed on the surface on multiple immune cell

types(26). Thus, the effects of IL-35 are likely to be exerted through a network of

interactions involving tumor and stromal cells.

To date, the evidence for B cell function in PDA has been scarce and seemingly

contradictory. Whereas, infiltration of CD20+ tumor-associated pan-B cell

population has been shown to correlate with better survival prognosis (27),

elevated levels of B cell activating factor (BAFF) have been reported to correlate

with metastatic propensity (28). These findings are in line with the increasing




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appreciation of the multifaceted role that B cells play in tumorigenesis. As part of

the adaptive immune system, B cells harbor the potential to mediate antitumor

responses by facilitating antigen presentation, effective priming of T cells and

anti-tumor antibody production (29, 30). On the other hand, B cells have been

shown to contribute to tumorigenesis by promoting alternative macrophage

activation (via deposition of immune complexes) and dampening T cell-mediated

anti-tumor response (B regulatory function) (14, 31). The findings described in

this study, along with those reported by Lee et al. and Gunderson et al. (12, 13),

illustrate that, depending on biological context, the pro-tumorigenic effects of B

cells could be mediated by distinct B cell populations. Thus, we have shown that

IL-35 producing B cells are required to support growth of early pancreatic

neoplasia. Gunderson et al. (12) have demonstrated that, in the setting of

advanced disease, the pro-tumorigenic role of B cells can be mediated by the

engagement of FcRϒ on tumor-associated macrophages resulting in their TH2

reprogramming. Lastly, Lee et al. (13) have reported an increase in B1b cells in

mouse neoplastic lesions that is further amplified upon loss of Hif1-alpha,

indicating that expansion of this B cell subset might be uniquely controlled by

oxygen sensing mechanisms. Functional dissection of how these various B cell-

dependent effector mechanisms are orchestrated would enable the full

delineation of the role of B cells in the development and maintenance of

pancreatic tumors.

MATERIALS AND METHODS




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Animal models

The LSL-KrasG12D, Pdx1-Cre and p48-Cre strains have been described

previously(3). C57BL/6 mice used for orthotopic injections and isolation of B cells

for adoptive transfers were obtained from The Charles River Laboratories. Both

female and male mice were used in studies. Randomization methods or

inclusion/exclusion criteria were not used to allocate animals to experimental

groups. Researchers were not blinded to the experimental groups while

conducting surgeries, as well as during data collection for orthotopic

transplantation into WT and μMT mice (due to very apparent spleen size

differences upon organ harvest and B cell differences in flow cytometry

experiments). Data collection for orthotopic transplantation into μMT mice

supplemented with B cells of various genotypes was conducted blindly.

Orthotopic implantation of PDEC was performed as described previously(3). In

the setting of orthotopic injection, GFP-KrasG12D-PDEC were injected at 1 x 106

cells/mouse pancreas and KPC cells were injected at 7.5 x 104 cells/mouse

pancreas. B cell-deficient μMT mice, IL10-/- and Il12a-/- animals were obtained

from Jackson Laboratories (strains #002288, 002251 and 002692 respectively).

All animal care and procedures were approved by the Institutional Animal Care

and Use Committee at NYU School of Medicine.

Isolation, Culture, and Infection of PDEC

Isolation, culture and adenoviral infection of PDEC were carried out as previously

described(32). KPC cell line (line 4662) was a kind gift from Dr. R. H. Vonderheide.




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Primary cell lines were not authenticated, and were tested for Mycoplasma

contamination every 4 months. To generate GFP-labeled PDEC lines, the cells

were infected with pLVTHM-GFP virus as described in(3). Briefly, lentivirus was

generated by transfecting HEK-293T cells with the vector, the packaging construct

(psPAX2), and the envelope plasmid (pMD2G). Supernatants containing viral

particles were collected over a period of 48 hours. Following final collection,

supernatants were filtered through a 0.45μm syringe filter and concentrated using

100MWCO Amicon Ultra centrifugal filters (Millipore).

Adoptive transfer of B cells

Spleens of WT C57Bl6 mice (2-3 month of age, Charles River Laboratories) were

mechanically dissociated, a single cell suspension was made in 1%FBS/PBS,

passed through a 70μm strainer (BD Falcon) and treated with RBC lysis buffer

(eBioscience). B cells were purified using CD45R-linked MACS beads (Miltenyi)

using LS columns according to manufacturer’s instructions. Enrichment of B cells

was confirmed by flow cytometry using FITC-CD19 (6D5, #115505, Biolegend).

Viability and numbers of purified B cells were assessed using Nexcelom

Cellometer Auto 2000 viability counter. Purified cells were then washed in cold

PBS and injected retro-orbitally into recipient mice (7x106 cells/mouse in 100μl

volume (WT, IL10-/- and IL12a-/- B cells) or 1.5x106 cells/mouse in 100μl volume

(CD19+CD1dhighCD5+ and CD19+CD1dlowCD5-).

Quantitative RT-PCR




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For RNA isolation, cells were enriched into B cell and non-B cell populations, as

well as immune and non-immune cells using CD45R-linked or CD45-linked

MACS beads (Miltenyi). Flow through fractions yielded non-B cells and non-

immune cells, respectively. Cells were then further processed by FACS:

CD19+CD1dhiCD5+ and CD19+CD1dlowCD5- B cells; CD45-CD140a+ fibroblasts

and CD45-CD140a- non-fibroblasts as well as CD45+CD11c+ dendritic cells were

FACS sorted using a 100μm nozzle from 3-6 month old KC mice pancreata (or

spleens for dendritic cells) into the lysing reagent Trizol (Invitrogen) and total

RNA was extracted as per manufacturer instructions (RNeasy mini kit, QIAGEN).

1μg of total RNA was reverse-transcribed using the Quantitect Reverse

Transcription kit (Qiagen). Subsequently specific transcripts were amplified by

SYBR Green PCR Master Mix (USB) using a Stratagene Mx 3005P

thermocycler. Where fold expression is specified, comparative CT method was

used to quantify gene expression. Where relative expression is specified,

standard curve method was used to quantify gene expression. Expression was

normalized to GAPDH.

Primers used for QPCR are as follows: GAPDH forward - CAC GGC AAA TTC

AAC GGC ACA GTC, reverse - ACC CGT TTG GCT CCA CCC TTC A; CXCL13

forward - GTA ACC ATT TGG CAC GAG GAT T, reverse - AAT GAG GCT CAG

CAC AGC AA; IL12a forward - CAT CGA TGA GCT GAT GCA GT, reverse -

CAG ATA GCC CAT CAC CCT GT; Ebi3 forward - TGC TCT TCC TGT CAC

TTG CC, reverse - CGG GAT ACC GAG AAG CAT GG; IL-10 forward - CAG

TAC AGC CGG GAA GAC AA, reverse - CCT GGG GCA TCA CTT CTA CC;




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IL12b forward - CAGCAAGTGGGCATGTGTTC, reverse -

TTGGGGGACTCTTCCATCCT; IL27 forward – TGTCCACAGCTTTGCTGAAT,

reverse – CCGAAGTGTGGTAGCGAGG.

Human Pancreas Specimens

For the purposes of analyzing B cell infiltration pattern and CXCL13 expression

pattern, we examined 10 samples containing PanIN lesions and 10 samples

containing PDAC lesions (20 samples total). Samples consisted of 5μm sections

that were cut from FFPE blocks provided by the Tissue Acquisition and

Biorepository Service (TABS) of the NYU School of Medicine. This study was

conducted in accordance with the Declaration of Helsinki; all samples were

anonymized prior to being transferred to the investigator’s laboratory and

therefore meet exempt human subject research criteria.

Histology and Immunohistochemistry

Mouse pancreata were fixed and processed for histology and

immunohistochemistry (IHC) as described previously(3). The IHC protocol was

modified to detect mouse and human CXCL13, where blocking was done in 1x

bovine free blocking solution (Vector) supplemented with 0.5% Tween-20, and

10% serum for 1 hour at room temperature, followed by incubation with the

primary antibody diluted in 1x bovine free blocking solution overnight at 40C.

Secondary biotinylated rabbit-anti-goat antibody (Vector) was diluted in 1x bovine

free blocking solution as well. The following primary antibodies were used: rabbit

anti-GFP (#2956S, Cell Signaling), rat anti-B220 (#BDB557390, Fisher), rabbit-




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anti-vimentin (#5741P, Cell Signaling), mouse-anti-CD20 (#555677, BD

Pharmingen), rabbit-anti-phospho Histone H3 (#06-570, Millipore), goat-anti-

mouse CXCL13 and goat-anti-human CXCL13 (#AF470 and # AF801, both from

R&D systems). At least 9 mice per experimental condition were analyzed for

GFP staining and 6 mice per condition were analyzed for pHH3 staining. Slides

were examined on a Nikon Eclipse 80i microscope.

Immunofluorescence

For paraffin sections: FFPE sections were deparaffinized and rehydrated,

permeabilized with TBS/0.1% Tween-20 and washed in PBS. Citrate buffer

antigen retrieval (10 mM sodium citrate/0.05% Tween-20 (pH 6.0)) was

performed in a microwave for 15 minutes. Blocking was performed in 10%

serum/1% BSA/0.5% Tween-20/PBS for 1 hour at room temperature. Primary

antibodies were diluted in 2% BSA/0.5% Tween-20/PBS and incubated on

sections overnight at 40C. Secondary antibodies (Alexa Fluor-labeled, Invitrogen)

were diluted in 2% BSA/PBS for 1 hr at room temperature. Sections were

washed with PBS and stained with DAPI. The following primary antibodies were

used: goat-anti-mouse CXCL13 (#AF470, R&D Systems), rabbit-anti-vimentin

(#5741P, Cell Signaling), mouse-anti-CD20 (#555677, BD Pharmingen), anti-

IL12a (#LS-B9481, LS Bio), anti-B220 (#BDB557390, Fisher), anti-IL-10 (#bs-

0698R, Bioss), anti-CD19 (#550284, BD Pharmingen). For frozen sections:

staining was performed as described in (3) using the following primary

antibodies: anti-IL12a ((#LS-B9481, LS Bio), anti-B220 ((#BDB557390, BD




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Pharmingen). Slides were examined using AxioVision v4.7 (Zeiss) software on a

Zeiss Axiovert 200M microscope.

Flow cytometry

Cellular suspensions from the tissues were prepared as described previously in 2.

The following antibodies were used: anti-CD19 (1D3, #45-0193-80, eBioscience),

anti-B220 (RA3-6B2, #RM2630, Life Technologies), anti-CD45 (104, #109825,

Biolegend), anti-CD1d (1B1, #123507, Biolegend), anti-CD140 (APA5, #135905,

Biolegend), anti-CD21 (7E9, #123419, Biolegend), anti-CD5 (53-7.3, #100607,

Biolegend), anti-AA4.1 (#17-5892, eBioscience), anti-CD138 (281-2, #142505,

Biolegend), anti-CD206 (C068C2, Biolegend), anti-CD86 (GL-1, Biolegend), anti-

F4-80 (BM8, Biolegend), anti-CD11b (M1-70, Biolegend). Dead cells were

excluded by staining with Propidium Iodine (Sigma-Aldrich) or Aqua Live/Dead

stain. Flow cytometry was performed on FACScalibur and LSRII II (BD

Biosciences) instruments at NYU School of Medicine Flow Cytometry Core

Facility and data was analyzed using FlowJo software.

Blockade of CXCL13

For CXCL13 neutralization experiments, anti-CXCL13 or a control IgG antibody

(both from R&D Systems), were injected at a concentration of 200 mg/mouse

(10). For experiments using KC animals, injections were performed twice per

week for one week. For experiments using orthotopically implanted animals, mice

were injected with the antibodies two days prior to implantation and then every 4

days post implantation for a total duration of two weeks.




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Statistical Analyses

Data are presented as means ± standard deviations (SD) or SEM, as indicated.

The experiments were repeated at a minimum of three times to demonstrate

reproducibility. In estimating orthotopic tumor size based on our previous data,

the standard deviation for our dependent variable is 2 units in wild type mice. We

would be interested in any differences between strains greater than 4 units.

Assuming equal variability and sample size in the two strains, a two-tailed alpha

of .05, and power of .80, we determined that we would need about 5-6 animals

per group to detect an effect as small as 0.5 SD units. Variance was similar

between the groups that were being statistically compared. Data were analyzed

by the Microsoft Excel built-in t test (unpaired, two-tailed) and results were

considered significant at p value < 0.05.

ACKNOWLEDGEMENTS

We thank L. J. Taylor for discussions and help with manuscript preparation, and

the members of Bar-Sagi lab for comments. Special thanks to Drs. George Miller,

David Tuveson, Ken Olive and Howard Crawford for their generous help with

mouse strains lost during hurricane Sandy.

GRANT SUPPORT

The FACS, Histopathology Cores, and Tissue Acquisition and Biorepository

Service of NYU School of Medicine are partially supported by the National




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Institutes of Health Grant 5 P30CA16087-31. This shared resource is partially

supported by the Cancer Center Support Grant, P30CA016087, at the Laura and

Isaac Perlmutter Cancer Center. This work was supported by the 2013

Pancreatic Cancer Action Network-AACR Inaugural Research Acceleration

Network Grant, supported by Tempur-Pedic in memory of Tim Miller, Grant

Number 13-90-25-VOND (D.B.-S. and Robert H. Vonderheide) and by the 2013

Pancreatic Cancer Action Network-AACR Pathway to Leadership Grant, Grant

Number 13-70-25-PYLA (Y.P.-G.).

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

AUTHOR CONTRIBUTIONS

YPG conducted most experimental studies and data analysis; RNA expression

analysis from FACS sorted cells was performed by YPG and SD; evaluation of

tumor growth with IL12a-/- B cells was performed by YPG and SD; JSH and SD

contributed to immunohistochemical staining and analysis of samples; CHH

selected and provided human tissue samples; MC and SK designed and facilitated

analysis of B cells; DBS directed all studies. YPG and DBS conceived the studies

and co-wrote the manuscript.

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FIGURE LEGENDS

Figure 1. B cells infiltrate mouse and human pancreatic neoplasia and

promote growth of KrasG12D-PDEC in vivo.

(A) Immunohistochemical detection of B cells in human (CD20 staining) and mouse

(B220 staining) in pancreata from hPanIN (20 patient samples), p48Cre (control, 5

mice), KC (10 mice), or KRasG12D-PDEC (9 mice) orthotopic lesions, as indicated.

Inset, B cells in the parenchyma of an adjacent tissue section detected by

immunofluorescence using anti-CD19 (green) and DAPI (blue). Representative

images are shown. Scale bars, 100μm.

(B) Hematoxylin and eosin (H&E) staining and immunohistochemical staining for

CD20, CXCL13 and vimentin in a representative sample of human pancreatic

cancer containing PanIN lesions (n=20). Inset, sections of human PanIN lesions

were stained by immunofluorescence (CXCL13, red; vimentin, green; and DAPI,

blue). Scale bars, 100μm; inset 7.5μm.

(C) Serial sections of a KC mouse pancreas were stained by immunohistochemistry

with CXCL13 or immunofluorescence (n=10; CXCL13, red; vimentin, green; and




24

DAPI, blue). A representative image is shown. Scale bars, 100μm and 12 μm

(inset).

(D) Expression of CXCL13 mRNA in cellular subsets isolated from pancreata of KC

mice. Error bars indicate SD. (n=6)

(E) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRasG12D-PDEC

implantation into WT or μMT mice were stained with H&E or anti-GFP antibody.

Where indicated, μMT mice were reconstituted with WT B cells 2 days prior to

orthotopic implantation. Representative images are shown. Scale bars, 100μm.

(F) Graph depicts quantification of the data in (E) and indicates the average fraction

of GFP+ signal per field of view (FOV; 10 FOV per animal; n=12 WT, n=14 μMT,

n=9 μMT+WT B cell animals).

Error bars indicate SD; P values were determined by Student’s t-test (unpaired,

two-tailed); p value: ***<0.001.

Figure 2. CD1dhighCD5+ B cells are expanded in pancreatic neoplasia and are

functionally important for sustaining growth of KrasG12D-PDEC in vivo.

(A) Quantification of flow cytometric analysis of plasma cells from spleens,

mesenteric lymph nodes (MLN), and pancreata of p48Cre (control) or KC mice. Cells

were analyzed for the presence of markers CD19, B220 and CD138 (n=5 p48Cre,

n=5 KC).

(B) Quantification of flow cytometric analysis of immune cells from pancreata of

p48Cre (control) mice, KC mice (2.5mo), or KRasG12D-PDEC orthotopic lesions (2

weeks), as indicated. After gating on CD19 and CD1d populations, cells were




25

analyzed for the presence of CD5 marker (n=8 p48Cre, n=8 KrasG12D-PDEC, n=8

KC).

(C) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRasG12D-PDEC

implantation into WT or μMT mice were stained with anti-GFP antibody. Where

indicated, μMT mice were reconstituted with WT CD19+CD1dhighCD5+ or with

CD19+CD1dlowCD5- 2 days prior to orthotopic implantation. Representative images

are shown. Scale bars, 100μm.

(D) Graph depicts quantification of the data from (C) indicating the average fraction

of GFP+ area per FOV of the implant (10 FOV per animal; n=12 WT, n=11 μMT,

n=9 μMT+ CD1dlowCD5-, n=9 μMT+ CD1dhighCD5+, animals).

(E) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRasG12D-PDEC


Where indicated, μMT mice were reconstituted with WT B cells or with IL10-/- B

cells 2 days prior to orthotopic implantation. Representative images are shown.

Scale bars, 100μm.

(F) Graph depicts quantification of the data from (E) indicating the average fraction

of GFP+ area per FOV of the implant (10 FOV per animal; n=14 WT, n=12 μMT,

n=12 μMT+WT B cell, n=12 μMT+IL10-/- B cell animals).

Error bars indicate SD; P values were determined by Student’s t-test (unpaired,

two-tailed); p value: *<0.05; **<0.01; ***<0.001; NS – not significant.

Figure 3. Expression of IL-35 by B cells is functionally important for

sustaining growth of KrasG12D-PDEC in vivo.




26

(A) Levels of IL12a mRNA in immune cells from spleen or pancreata of p48Cre

(control) or KC mice were assessed by quantitative RT-PCR (n = 9 p48Cre, n=9 KC).

(B) Levels of IL12a mRNA in CD19+CD1dhighCD5+ and CD19+CD1dlowCD5- sub-

populations of B cells sorted from pancreata of KC mice were assessed by

quantitative RT-PCR (n=9 KC).

(C) Levels of Ebi3 mRNA in B cells and non-B cells from pancreata of KC mice

were assessed by quantitative RT-PCR (n=9 KC).

(D) Levels of Ebi3 mRNA in CD19+CD1dhighCD5+ and CD19+CD1dlowCD5- sub-

populations of B cells sorted from pancreata of KC mice were assessed by

quantitative RT-PCR (n=9 KC).

(E) Immunofluorescence staining for p35 and CD20 in samples of human

pancreatic cancer containing PanIN lesions. Scale bars, 10μm (top) and 20μm

(bottom). Two independent fields of view are shown.

(F) Immunofluorescence staining for p35 and B220 in samples of KC pancreata.

Scale bars, 20μm. Two independent fields of view are shown.

(G) Sections from orthotopic pancreatic grafts 2 weeks after GFP-KRasG12D-PDEC


Where indicated, μMT mice were reconstituted with WT B cells or with IL12a-/- B

cells 2 days prior to orthotopic implantation. Representative images are shown.

Scale bars, 100μm.

(H) Graph depicts quantification of the data from (G) indicating the average fraction

of GFP+ area per FOV of the implant (10 FOV per animal; n = 9 WT, n=9 μMT, n= 9

μMT+WT B cell, n=9 μMT+IL12a-/- B cell animals).




27

(I) Immunohistochemical staining for phospho-Histone H3 of GFP-KRasG12D-PDEC

implanted into mice as described in (G) above. Representative images are shown.

Scale bars, 50μm.

(J) Graph depicts quantification of the data in (I) and indicates the fraction of

phospho-Histone H3+ signal in epithelial cells (10 FOV per animal; n=6 WT, n=6

μMT, n=6 μMT+WT B cell, n=6 μMT+IL12a-/- B cell animals).

Error bars indicate SEM in A, SD in B-D, H, J; P values were determined by

Student’s t-test (unpaired, two-tailed); p value: *<0.05; **<0.01; ***<0.001.




Figure 1

A control KRasG12D-PDEC KC hPanIN

Mouse Human

CD20 B220 B220 B220

D

CX

CL13 m

RN

A e

xpre

ssio

n

1.2

1

0.8

0.6

0.4

0.2

CD45+ CD11c+

B H&E CD20 CXCL13 vimentin

Human

C

F 35

30

25

20

15

10

5

***

μMT WT μMT

+WT B cells

***

GF

P +

sig

nal (p

erc

ent/

FO

V)

E WT μMT

μMT

+ WT B cells

H&

E

GF

P

DAPI

CXCL13

Vimentin

CXCL13

CXCL13

Vimentin

DAPI




GF

P +

sig

nal (p

erc

ent/

FO

V)

NS

WT μMT CD1dlow

CD5-

CD1dhigh

CD5+

30

25

20

15

10

μMT

5

D

**

* **

*

B

Control KC KrasG12D

PDEC

10

5

15

20

%C

D19

hig

hC

D1

dh

igh c

ells

* ***

Figure 2

GF

P +

sig

nal (p

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FO

V)

F

25

20

15

10

5

μMT WT +WT

B cells

+IL10-/-

B cells

μMT

*** *** ***

E WT μMT

μMT

+ WT B cells

μMT

+ IL10-/- B cells

H&

E

GF

P

C WT μMT

μMT +

CD1dlowCD5-

μMT +

CD1dhighCD5+

A %

CD

19

low

/-B

22

0lo

wC

D1

38

+ c

ells

*

control KC

Spleen

control KC

MLN

control KC

Pancreas

0.5

1

1.5

2

2.5

3




Figure 3

CD20

DAPI

E Human PanIN

p35

CD20

DAPI

p35

DAPI

A

non-B

cells

IL12

a e

xp

ressio

n f

old

ch

an

ge 10

8

6

4

2

B cells

C

B cells non-

B cells

Ebi3

expre

ssio

n f

old

ch

an

ge 3.5

3

2.5

2

1.5

1

0.5

F

B220

DAPI

Mouse PanIN

p35

B220

DAPI

p35

DAPI

D 25

20

15

10

5

Ebi3

expre

ssio

n f

old

ch

an

ge

**

B

IL12

a e

xp

ressio

n f

old

ch

an

ge 7

6

5

4

3

2

1

** ** ** spleen pancreas

Control Control KC KC CD1dlow

CD5-

CD1dhigh

CD5+

CD1dlow

CD5-

CD1dhigh

CD5+

WT μMT

μMT

+ WT B cells

μMT

+ IL12a-/- B cells

H&

E

GF

P

30

25

20

15

10

5

μMT WT +WT

B cells

+IL12a-/-

B cells

μMT

G H

J

% p

-HH

3+ e

pithelia

l cells

14

12

10

8

6

4

μMT WT +WT

B cells

+IL12a-/-

B cells

μMT

I

pH

H3

GF

P+ s

ignal (p

erc

ent/

FO

V)

*** ***

*** ***

2

* *

* *




Published OnlineFirst December 29, 2015.Cancer Discov Yuliya Pylayeva-Gupta, Shipra Das, Jesse S. Handler, et al. neoplasiaIL-35 producing B cells promote the development of pancreatic

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Yuliya Pylayeva-Gupta , Shipra Das , Jesse S. …...2015/12/28 · Yuliya Pylayeva-Gupta1,3, Shipra Das1, Jesse S. Handler1, Cristina H. Hajdu2, Maryaline Coffre2, Sergei Koralov2,