the presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/imm.12417 This article is protected by copyright. All rights reserved.

Received Date : 23-Jun-2014

Revised Date : 17-Oct-2014

Accepted Date : 19-Oct-2014

Article type : Original Article

Title:

The presence of interleukin-27 during monocyte-derived dendritic cell differentiation

promotes improved antigen processing and stimulation of T cells

Short title:

The influence of IL-27 during DC differentiation

Authors:

Joo-Yong Jungǂ, Lawton L. Roberts*, and Cory M. Robinson§

Author’s affiliations:

Department of Biology, Briar Cliff Universityǂ, Department of Pathology, Microbiology, and

Immunology, University of South Carolina School of Medicine*, Biomedical Sciences

Department, West Virginia School of Osteopathic Medicine§

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This article is protected by copyright. All rights reserved.

§ Corresponding author: Cory M. Robinson

Address: Cory M. Robinson, Ph.D., Biomedical Sciences Department, West Virginia School

of Osteopathic Medicine, 400 North Lee Street, Lewisburg, WV 24901.

Telephone: 304-647-6370

Email: [email protected]

Keywords: Interleukin-27, dendritic cells (DCs), antigen presenting cell

Summary

Dendritic cells (DCs) are potent antigen presentation cells necessary to establish effective

adaptive immune responses. The cytokine environment that exists at the time of DC

differentiation may be an important but often ignored determinant in the phenotypic and

functional properties of DCs. Interleukin (IL)-27 is a unique cytokine that has both

inflammatory and immune suppressive activity. While it can both promote and oppose

activity of different T cell subsets, mostly anti-inflammatory activity has been described

toward macrophages and DCs. However, the specific effect of IL-27 during DC

differentiation and how that may change the nature of the antigen presenting cell has not been

investigated. In this report, we show that IL-27 treatment during monocyte-derived DC

differentiation enhanced the ability to process antigens and stimulate T cell activity. DCs

differentiated in the presence of IL-27 showed enhanced acidification of latex bead-

containing phagosomes that was consistent with elevated expression of vacuolar-ATPases.

This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the

levels of MHC class II surface expression were higher in DCs differentiated in the presence

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of IL-27. IL-12 production was also significantly increased during S. aureus infection of IL-

27-differentiated DCs. The net effect of these activities was enhanced CD4+ T cell

proliferation and Th1 cytokine production. These findings are important to a wide number of

immunological contexts and should be considered in the development of future vaccines.

Introduction

Dendritic cells (DCs) are professional antigen presenting cells that can initiate and modulate

immune responses in a variety of ways. DCs undergo unique developmental stages to

effectively direct antigen specific immune responses (1). In the immature stage, DCs are

specialized in antigen capture and consequently express high levels of intracellular MHC

class II while maintaining high endocytic and phagocytic capacity (2). DCs at this stage

express chemokine receptors such as CCR1, CCR5, and CCR6 that direct them to sites of

inflammation (2). After capturing antigen, DCs upregulate CCR7 that directs migration to

secondary lymphoid organs (3). While migrating, DCs undergo maturation that limits their

ability to capture antigen, while increasing the ability to process and present antigens. DCs at

this stage express high levels of MHC class II at the cell surface as well as additional cell

adhesion and T cell costimulatory molecules such as CD40, ICAM-1 (CD54), LFA-3 (CD58),

CD80, CD83, and CD86 (2). This repertoire of surface molecules on mature DCs promotes

interaction with lymphocytes and antigen presentation through MHC class II that allows for

the selection of antigen-specific lymphocytes (2).

Interleukin (IL)-27 is produced by antigen presenting cells in response to a variety of

activation stimuli, notably microbial-derived products (4-9). IL-27 was originally described

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as a soluble factor that promotes Th1 differentiation (10). However, IL-27 also negatively

regulates these same cells (4, 11). IL-27 is also known to inhibit differentiation of Th17 cells

and production of IL-17 by inducing Tr-1 cells that produce high levels of IL-10 (12, 13).

Similarly, immunosuppressive activity of IL-27 has been described toward a number of other

immune cell types, including B cells and myeloid cells such as macrophages and DCs. (6-9,

14-20). IL-27 is expressed by M. tuberculosis-infected human macrophages (6- 9). Recently,

we published that IL-27 negatively regulates phagosomal acidification by decreasing the

expression of vacuolar ATPases in human macrophages (9, 19). IL-27 treatment of

monocyte-derived mature DCs is immunosuppressive by increasing B7-H1 and decreasing

CD80, CD83, and CD86 (21). B7-H1 is a member of the B7 family that does not interact

with CD28, CTLA-4, or ICOS on T cells and opposes inflammatory T cell activity (22).

Similarly, DCs from IL-27 receptor-deficient mice exhibit enhanced antigen presenting

function that included prolonged expression of CD80, CD86, and enhanced IL-12 production

that increased priming of CD4+ T cells to produce IFN-γ (16).

The cytokine microenvironment during cellular differentiation may significantly

impact the phenotypic and functional characteristics of DCs. Pro-inflammatory properties of

IL-27 toward monocytes have been reported (23, 24). IL-27 promoted enhancement of MHC

class I and II expression in THP-1 cells (23). In primary human monocytes, IL-27 activated

STAT-1-dependent inflammatory genes, augmented TLR responses, and inhibited IL-10

production (25). Here we specifically evaluated the effect of IL-27 on the differentiation of

primary human DCs from monocyte precursors. IL-27-mediated DC differentiation is

inflammatory and increases lysosomal acidification, MHC class II expression, the production

of IL-12, and bacterial control. This is relevant and important in both acute and chronic

infections when monocytes are recruited to sites of inflammation in tissues and subsequently

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receive signals for differentiation. Consequently, these results should be considered in the

development of vaccines and immunotherapies.

Materials and Methods

Cell culture

Human buffy coats were purchased from the New York Blood Center (New York, NY).

Eligible donors were 16 years of age or older, at least 110 pounds, and in good physical

health. The donor samples were anonymous and de-identified. Peripheral blood

mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll gradient centrifugation.

Monocytes were then isolated from PBMCs by Optiprep (Sigma-Aldrich) gradient

centrifugation as described previously (26). Monocytes were adhered to plastic 60 mm

culture dishes in serum-free DMEM. Media was replaced with DC differentiation media

(RPMI supplemented with 2 mM glutamine, 0.1 mM non-essential amino acid, 1 mM sodium

pyruvate, 0.05 mM 2-mercaptoethanol, 25 mM HEPES, recombinant human IL-4 [1600

U/106 cells, Gemini], GM-CSF [290 U/106 cells, Gemini], and 10% human serum) and

incubated at 37°C with 5% CO2 for 7 days. For some cultures, IL-27 was included in DC

differentiation media on day zero. For initial experiments a range of IL-27 concentrations up

to 100 ng/mL was used and it was determined that 40 ng/mL was sufficient for results

reported here. Loosely adherent DCs were removed from the culture dish with PBS that

contained 5 mM EDTA and 4 mg/mL lidocaine. The cells were washed with PBS and plated

onto new culture dishes in culture medium (RPMI supplemented with 2 mM glutamine, 0.1

mM non-essential amino acid, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 25 mM

HEPES, and 10% human serum). These cells are routinely >95% CD11chigh, MHC IIhigh,

CD86+, CD80+, CD1b+, CD40high, DC-SIGN+ (CD 209), and CD14- .

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Bacterial infection and bafilomycin treatment

Staphylococcus aureus RN6390 was kindly provided by Dr. Mark Hart (University of North

Texas Health Science Center). The bacteria were grown overnight in Tryptic Soy broth at

37°C and washed in PBS. The bacteria were adjusted to 1×108 (colony forming units)

CFU/ml using a spectrophotometer (Optical density [OD]600 nm=0.4). DCs were left

untreated or pretreated with bafilomycin (100 nM, Sigma) for four hours to block V-ATPase-

mediated lysosomal acidification as described previously (9). Next, DCs were infected at a

multiplicity of infection (MOI) of ~10 for 1 h. Gentamycin (10 μg/ml) was then added to the

infected cultures to kill extracellular staphylococci and the infection was allowed to proceed

for an additional 2, 12, or 24 h. To enumerate intracellular bacteria, DCs were permeabilized

with a 0.1% solution of saponin in PBS followed by standard serial dilution plating.

Analysis of lysosomal acidification and immunolabeling

Human DCs cultured in 24-well plates were analyzed for the level of lysosomal acidification.

In the last hour of infection, culture supernatants were replaced with medium that contained

Lysotracker DND-99 Red (Life Technologies) (100 nM). The slides were examined using a

Zeiss Meta 510 laser confocal microscope with a plan-Apochromat 63X objective lens. A

total of 10 fields containing 5-10 DCs per field were examined in each experiment. The

mean fluorescent intensity (MFI) for each DC was calculated using Image J software. Each

cell from the image was selected and histogram analysis was performed. For immunostaining,

mouse monoclonal antibodies for V1-ATPase H (sc-166227, Santa Cruz Biotechnology)

were visualized with anti-mouse-Alexafluor 568-conjugated secondary antibody.

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Quantitative PCR

Human DCs (1.5x105/well) cultured in 24-well dishes were subjected to RNA isolation. At

appropriate time points, the media was removed from cultures, the cells were lysed with

PureZol® (Bio-Rad), and RNA was isolated according to commercial product protocol. First

strand cDNA synthesis was performed using iScript™ cDNA synthesis reagents (Bio-Rad)

according to protocol. Primers were synthesized by Integrated DNA Technologies, Inc. The

following primer sets were used for amplification of HLA-DR or IL-12 transcripts with

SsoFast™ EvaGreen® supermix (Bio-Rad): IL-12 p35 forward; 5’-atgctccagaaggccagac-3’

reverse; 5’-tctggaatttaggcaactctca-3’ IL-12 p40 forward; cctggagaaatggtggtcct-3’ reverse; 5’-

gcttagaacctcgcctcctt-3’ HLA-DR forward; 5’-agcagtcatcttcagcat-3’ reverse; 5’-

atgttagagtacggagcaat-3’ GAPDH forward; 5’-cagccgcatcttcttttg-3’ reverse; 5’-

gcaacaatatccactttacca-3’. Gene expression was normalized to that of GAPDH, expressed

relative to untreated controls using the 2-ΔΔCt method, and log2 transformed.

Immunoblot analysis

Whole cell lysates were prepared from human DCs (1.5x105/well) cultured in 24-well dishes.

Some of the cultures were infected with S. aureus as described above. PBS supplemented

with 1% Tx-100 (40 µl) was applied to each sample and lysates collected by scraping. They

were subsequently sonicated briefly and then stored at 4°C. Equal amounts of cell lysates

were separated on SDS-PAGE gels and transferred to nitrocellulose by standard techniques.

Primary antibodies for V-ATPase H, actin, or all forms of cathepsin D were revealed with

horse radish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies. ECL

substrate (Amersham Biosciences) was applied to visualize proteins.

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ELISA analysis

Human DCs were cultivated as indicated above. Following the indicated treatment,

supernatants were collected at indicated time points for analysis of IL-12p70 (R&D Systems),

IL-2 (Ebioscience), or IFN-γ (R&D Systems) concentrations. Standard curves were

performed in parallel.

Flow Cytometry

DCs were harvested and phenotypic analysis performed as previously described (27). Briefly,

DCs were suspended in 0.1 mL of staining buffer (0.5% BSA in PBS) and Fc receptor

blocked for 20 min. Next, cells were immunolabeled with monoclonal antibodies against the

indicated molecules for 30 min and subsequently fixed with 0.5 mL 4% PBS-buffered

paraformaldehyde. At least 5,000 cells were analyzed for each sample on a FC 500

(Beckman Coulter) flow cytometer.

T cell proliferation assays

Monocyte-derived DCs differentiated in the presence or absence of IL-27 were harvested (as

above) and seeded (5 x 104) on a 96-well plate overnight in culture medium. The next day,

DCs were infected with an overnight culture of S. aureus at a MOI ~ 10 for 1 h. The media

was then removed and replaced with medium containing gentamycin (10 μg/mL) for 3 h.

Next, CD4+ T-cells were isolated from monocyte-depleted fractions by immunomagnetic

selection using a human CD4+ T cell isolation kit (Miltenyi Biotec) according to the

instructions provided by the manufacturer. The resulting CD4+ T cells were stained with

CFSE (10 μM, Biolegend) in culture medium for 10 min at room temperature. Staining was

quenched by washing once with 5 mL of room temperature FBS. Next, 2 x 105 CFSE-stained

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T cells in culture medium were antibody stimulated with 10 μg/mL of plate-bound anti-CD3

(BD Pharmingen) and 1 μg/mL soluble anti-CD28 (BD Pharmingen), left unstimulated, or

cultured with syngeneic DCs. Cells were incubated at 37o C with 5% CO2 for 5 days.

Aliquots for analysis of secreted IL-2 and IFN-γ were collected on days 1 and 5 of five

independent experiments done with separate donors. In addition, on day 5 of three

independent experiments described above, the cells were harvested and analyzed by flow

cytometry as above. T cell proliferation was determined by CFSE dilution. CFSE-stained T

cells were distinguishable from DCs. To exclude DCs from the analysis, a gate was then

established for the population that represented CFSE-stained T cells (dot plot quadrant A4,

Fig. 5 A and C). To quantify the percent T cell proliferation, we established another gate

(histogram region E, see Fig. 5 panels D-G) using the antibody controls (Fig. 5D and E) that

represents CFSE dilution indicative of proliferation. Accordingly, the percentage of gated T

cells within region E was quantified. Ten-thousand cells were analyzed for each duplicate

treatment.

Results

DCs differentiated in the presence of IL-27 exhibit enhanced phagosomal acidification

IL-27 exhibits anti-inflammatory activity towards established macrophages and DCs (4, 19).

However, inflammatory activity has been associated with IL-27 toward monocytes. Since

macrophages and DCs can differentiate from monocyte precursors, we wanted to understand

if the presence of IL-27 during differentiation from a monocyte would lead to development of

a suppressive or inflammatory phenotype. To address this, monocytes were cultured with or

without IL-27 during 7 days of differentiation to DCs. An important function of DC biology

is the uptake of antigen and processing for presentation to lymphocytes. After 7 days of

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differentiation, the DCs were subjected to treatment with fluorescently labeled latex beads (2

μm) for 6 h. We did not observe a difference in the uptake of latex beads by DCs

differentiated in the presence or absence of IL-27 (not shown). Next, we evaluated the acidic

nature of the compartment surrounding the latex beads that is important for antigen

processing. Following uptake of latex beads, lysotracker (100 nM) was added to the DC

cultures that were subsequently incubated for an additional 2 h. Surprisingly, DCs

differentiated in the presence of IL-27 demonstrated enhanced acidification of the latex bead

compartment (LBC) compared to DCs differentiated without IL-27 (Fig. 1A). The level of

increased acidification was approximately 1.7 fold (Fig. 1B). Phagosomal acidification is

mediated by the expression and localization of V-ATPases that allow for a decrease in pH

from 6.5 to 5.0 (19). This acidic environment inhibits the growth of microorganisms and

further enhances the recruitment and activity of hydrolytic enzymes allowing for enhanced

antigen processing. Therefore, we examined V-ATPase localization (Fig. 1C). V-ATPase

was strongly recruited to the LBC in DCs differentiated with IL-27 and the percent

colocalization was approximately two–fold greater (Fig. 1C and D). This is likely to be

mediated, at least in part, by the increased expression of V-ATPases since signal intensity of

V-ATPases was dramatically increased by 6 h (Fig. 1C, and E). This was further

substantiated by immunoblot analysis in which V-ATPase expression was increased in DCs

that received IL-27 by approximately 1.5-fold (Fig. 1F and G). Cathepsin D is a soluble

lysosomal endopeptidase synthesized in the endoplasmic reticulum as pre-procathepsin D and

following the removal of the signal peptide, the 52 kDa procathepsin D is localized to

endosomes, phagosomes, or lysosomes (28). Phagosomal acidification allows for cleavage of

the amino-terminal propeptide yielding a 48 kDa single chain active enzyme that can undergo

additional cleavage to generate a mature lysosomal protease (24 to 30 kDa) (28). We

investigated whether or not there was a difference in processing of cathepsin D to the mature

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form in DCs differentiated in the presence of IL-27. Immunoblot analysis using an antibody

that recognizes all three forms of cathepsin D, demonstrated that DCs differentiated in the

presence of IL-27 exhibited increased accumulation of the mature form of Cathepsin D (Fig.

1H). Overall, this data suggests that the presence of IL-27 during DC differentiation increases

the potential to process antigen.

DC differentiation in the presence of IL-27 promotes bacterial clearance in human DCs

Acidification of phagosomes and activation of cathepsin D is critical for DC-mediated control

of bacterial pathogens (29, 30). Since DCs differentiated with IL-27 exhibited increased

acidification of phagosomes and accumulation of active cathepsin D, we hypothesized that

these DCs may clear bacteria more efficiently. To address this possibility, we infected DCs

with S. aureus and enumerated survival. This bacterium was chosen for two reasons.

Recently it has been shown that S. aureus is capable of surviving and multiplying within DCs

(31). Secondly, because this bacterium is a common commensal, the pool of blood donors

used in our studies likely contains circulating lymphocytes with staphylococcal antigen

specificity important for later experiments. To directly address the requirement for V-

ATPase-mediated lysosomal acidification on bacterial clearance, DCs differentiated in the

presence or absence of IL-27 were pretreated with or without bafilomycin (100 nM) for 4 h

and then infected with S. aureus (MOI=10). After 1 h, gentamycin (10 µg/ml) was added to

kill extracellular bacteria that were not internalized. The cultures were incubated for an

additional 2, 12, or 24 h. Consistent with the enhanced lysosomal acidification and cathepsin

D activity, bacteria were cleared by 12 h in DCs differentiated in the presence of IL-27 (Fig.

2). This bacterial clearance was inhibited by bafilomycin treatment demonstrating a

dependence on lysosomal acidification. This data further suggests an influence of IL-27

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during the differentiation of monocyte-derived DCs that promotes antibacterial activity and

antigen processing capability.

DC differentiation in the presence of IL-27 enhances expression of MHC class II

Since phagosomal acidification that is important for antigen processing was more efficient in

DCs differentiated in the presence of IL-27, we wanted to investigate whether or not these

DCs would be better antigen presenters. To address this, we investigated the cell surface

expression of HLA-DR in DCs differentiated in the presence and absence of IL-27. HLA-DR

was more highly expressed at the cell surface in DCs differentiated with IL-27 (Fig. 3A). S.

aureus infection further increased surface levels on both DC treatment groups, but a similar

trend was maintained (Fig. 3A). Quantitative analysis of mean fluorescent intensity (MFI)

showed an approximate two-fold increase of HLA-DR surface expression on DCs

differentiated in the presence of IL-27 (Fig. 3B). This result was at least partly influenced by

enhanced HLA-DR gene expression in DCs differentiated in the presence of IL-27 (Fig. 3C).

Dendritic cells differentiated in the presence of IL-27 upregulate cell surface molecules

that facilitate activation of T cells.

Since we observed an increase in MHC class II expression when DCs were differentiated in

the presence of IL-27, we wanted to determine if the presence of IL-27 would also augment

expression of cell surface molecules known to facilitate activation of lymphocytes. To do

this, DCs were differentiated as before, harvested, and immunolabeled for a number of cell

surface molecules, and analyzed by flow cytometry. There was no significant labeling with

isotype control antibodies; this was not altered by IL-27 treatment. Consistent with increased

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MHC class II expression, numerous surface molecules were upregulated by the addition of

IL-27 (Table 1). Specifically, we observed an approximate 50% and 37% increase in the

MFI of DC-SIGN and CD40, respectively (Table 1). ICAM-1 (CD54) expression was

increased although this did not achieve statistical significance (Table 1). Additionally, the

integrin subunits CD18 and CD11b (collectively Mac-1; CR3) were also significantly

increased at the cell surface in the presence of IL-27 over four independent blood donors

(Table 1). The expression of T cell costimulatory molecule CD86 was unaffected by the

addition of IL-27 (data not shown).

Interleukin (IL)-12 production was increased in DCs differentiated in the presence of

IL-27

IL-12 production by DCs is important for effective initiation of Th1 responses (32). DCs

infected by S. aureus secrete IL-12 (31). Since we observed enhanced lysosomal

acidification, MHC class II expression, and surface levels of molecules important for

interactions with lymphocytes, we examined IL-12 production in DCs differentiated in the

presence or absence of IL-27. To address this, DCs were infected with S. aureus for 24 h and

then RNA was harvested for analysis of IL-12p35 and p40 gene expression. IL-12 p35 and

p40 gene was highly expressed in infected DCs that received IL-27 (Fig. 4A). However,

secreted IL-12p70 was only detected in culture supernatants of S. aureus-infected DCs that

were differentiated in the presence of IL-27 (Fig. 4B). We also measured levels of secreted

IL-23 that incorporates the p40 subunit, as well as IL-6, but did not observe significant

changes between treatment groups (not shown). Collectively, this data further support the

idea that DCs differentiated in the presence of IL-27 may be stronger APCs.

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Dendritic cells differentiated in the presence of IL-27 are more potent stimulators of T

cell activity

Given the augmentation of MHC class II, numerous cell surface molecules involved in T cell

interactions, and enhanced IL-12 production by the addition of IL-27 during DC

differentiation, we wanted to test the functional impact on the ability to stimulate T cells.

This was done with T cell proliferation assays that incorporated syngeneic, primary human T

cells and DCs. Considering common exposure to S. aureus in our donor pool, it is reasonable

to assume the presence of circulating antigen-specific T cells. CD4+ T cells were CFSE-

stained and co-cultured with DCs for 5 days. T cell proliferation was determined by analysis

of CFSE dilution by flow cytometry. CFSE-stained T cells were distinguished from DCs on

the basis of CFSE staining (Fig. 5 A-E). Thus, to exclude DCs from the analysis and to

quantify T cell proliferation, we gated on the CFSE-stained T cell quadrant (quadrant A4, Fig.

5C) to create histograms that express the level of T cell proliferation (Fig. 5 D-G). T cells

were either left untreated, stimulated with anti-CD3 and anti-CD28 (Ab stimulated), or

cultured with S. aureus-infected DCs that were differentiated in the absence (Med DC, Fig.

5F) or presence (IL-27 DC, Fig. 5G) of IL-27. As shown in Figure 5E, antibody stimulation

of T cells resulted in several proliferative peaks (region E), and this region was used to

quantify percent T cell proliferation. This analysis demonstrated that DCs differentiated in

the presence of IL-27 and then infected with S. aureus stimulated a 5% increase in T cell

proliferation compared to DCs not differentiated with IL- 27 (Fig. 5 F-H). This result was

consistent over three independent blood donors and reached statistical significance (p= 0.023).

Even more striking was the difference in secreted cytokine levels. T cells stimulated by

infected DCs differentiated in the presence of IL-27 increased the early production of IL-2

(Fig. 5I) and IFN-γ (Fig. 5J) over time compared to normally differentiated DCs. IL-2 levels

were increased significantly at day 1 (Fig. 5K; p= 0.035 over 5 independent donors.

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Similarly, IFN-γ secretion elicited by IL-27 differentiated DCs was significantly increased at

day 5 (Fig. 5L; p= 0.04) over 5 independent donors. The early consumption of IL-2 during

proliferative events is inversely correlated with enhanced IFN-γ production. The kinetics of

this reciprocal cytokine pattern is consistent with enhanced T cell stimulation, and further

substantiates that monocyte-derived DCs differentiated in the presence of IL-27 are stronger

stimulators of T cell activity.

Discussion

The cytokine milieu is an essential determinant of effective immunity as it shapes the

development of cellular phenotypes and the nature of the response. Consequently, we wanted

to determine the impact of IL-27 on monocyte-derived DC differentiation. Since monocytes

are recruited to sites of inflammation where IL-27 is produced, this is a relevant and

important consideration in the development of DCs that are essential in the establishment and

orchestration of immune responses. To the best of our knowledge, we are the first to

examine the effects of IL-27 during the differentiation of monocyte-derived DCs. IL-27 is

known to possess dual immunological functions (11). It is well established that IL-27 exerts

anti-inflammatory activity towards macrophages (4, 6, 7, 8, 9, 11, 19). Several reports have

documented anti-inflammatory properties of IL-27 on terminally differentiated DCs, both

murine and human (16, 21). In murine DCs, using allogeneic T cell proliferation assays,

Wang found that DCs deficient for the IL-27 receptor α chain (IL-27Rα) induced greater

levels of IFN-γ and proliferation of CD4+ T cells compared to wild type DCs (16). However,

they saw no differences in IL-2 production. This suggests that IL-27R signaling suppresses

DC function as an APC. In another report, Karakhvona and colleagues found that treatment

of human, monocyte-derived DCs with IL-27 induced expression of B7-H1 and reduced their

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ability to stimulate allogeneic T cells (21). Similar to our findings, they also found no effect

of IL-27 on the T cell costimulatory molecules CD80 and CD86. In contrast, we report a

response to IL-27 during differentiation that drives monocyte-derived DCs to be more potent

antigen processing, presenting, and T cell-stimulating cells.

The acidic nature of the phagosome is important for antigen processing and can

generally reflect a suppressive or inflammatory phenotype of antigen presenting cells. The

LBC is a suitable model to study phagosomal acidification that is important for antigen

processing. We previously showed that treatment of terminally differentiated macrophages

with IL-27 significantly decreased acidification of the LBC (19). This de-acidification

correlated with reduced expression of V-ATPases and limited maturation of cathepsin D to

the active form (19). Surprisingly, in DCs differentiated in the presence of IL-27, the LBC

was significantly more acidified within 6 h. This acidification was consistent with elevated

expression of V-ATPases (Fig. 1). The number of S. aureus recovered from DCs was

significantly decreased in those that received IL-27 (Fig. 2), and this was reversed by treating

DCs with bafilomycin (a V-ATPase inhibitor). This suggests that the bacteria were internally

processed via the phagolysosomal pathway, and also demonstrates that IL-27 enhanced DC-

mediated clearance of bacteria and the cellular machinery necessary to process antigens.

These results are in contrast to the effects of IL-27 on mature macrophages that we have

observed and reported (9, 19). However, when macrophages were differentiated from

monocytes in the presence of IL-27, we did not observe an influence on phagosomal

acidification (data not shown). These studies suggest that the timing of IL-27 signaling is

important to the functionality of the cell, but also continue to emphasize the divergent nature

of IL-27 toward various cell types (11). That both macrophages and DCs can develop from

monocyte precursors suggests that the nature of the differentiation signal and interaction with

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IL-27 is important. Collectively, our data demonstrate that IL-27 elicits differential effects on

different cell types, and influences the cellular outcomes following differentiation.

In addition to the antigen processing capability, MHC class II expression on the cell

surface reflective of antigen presentation was increased in DCs differentiated in the presence

of IL-27 (Fig. 3). This is consistent with the reported proinflammatory nature of IL-27

toward monocytes that included increases in MHC class I and II and costimulatory molecules

(23, 24). In this report, we specifically demonstrate that when IL-27 is added during the

differentiation of monocytes to DCs, there is an increase in cell surface molecules that

mediate important DC biological functions, particularly T cell activation (Fig. 4).

Recognition of DC presented antigen to T cells requires the migration of DCs from antigen

capturing regions through lymphatic endothelia to draining lymph nodes, followed by the

formation of an immunological synapse; both of which are carefully orchestrated by

numerous cell-cell interactions. Of the molecules facilitating these processes, DC-SIGN has

been shown to mediate DC adhesion, antigen uptake, and activation of resting T cells (33-35).

In a similar body of work, anti-DC-SIGN antibodies inhibited DC-induced proliferation of

resting T cells (34). This suggests a fundamental role for DC-SIGN in mediating and

establishing DC-induced immune responses. In line with this, we show a significant increase

in cell surface expression of DC-SIGN on DCs differentiated in the presence of IL-27.

Engering and colleagues have reported that DC-SIGN also functions as an antigen receptor

that efficiently targets internalized antigens to lysosomes for processing and presentation to T

cells (35). We do not specifically present data to address this link with antigen processing,

but increased expression of DC-SIGN may function similarly in our system. We also

observed a significant increase in surface levels of CD40 and Mac-1 (CD18/CD11b; CR3) on

DCs differentiated in the presence of IL-27 (Table. 1). Ligation of CD40 on DCs by its

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cognate receptor CD40L primes DCs to become more efficient APCs. Several groups have

shown that CD40:CD40L interaction favors upregulation of adhesion and costimulatory

molecules by DCs with enhanced cytokine production, including IL-12 (36, 37). The integrin

Mac-1 functions as a cell adhesion receptor and in phagocytosis of opsonized and non-

opsonized antigens, respectively (38). Concomitant with increased expression of surface

molecules that contribute to APC function (Table 1), enhanced IL-12 production (Fig. 4)

suggests an important effect of IL-27 on DC differentiation that ultimately manifests as an

APC phenotype better suited to prime T cells and promote IFN-γ production. We also

examined levels of IL-23 that shares a common p40 subunit with IL-12, as well as IL-6, and

found no difference between DCs differentiated in the presence or absence of IL-27 (not

shown). To determine the functional significance of IL-27 on DC differentiation, we tested

whether these DCs would exhibit an enhanced ability to stimulate T cell proliferation using

syngeneic T cell proliferation assays. As expected, IL-27 differentiated DCs were more

potent T cell stimulators, as determined by CFSE dilution and Th1 cytokine production (Fig.

5) in response to S. aureus infection. We report a significant increase (p= 0.023) in T cell

proliferation in the presence of DCs that were differentiated in the presence of IL-27.

Although it was expected that the donor pool would have S. aureus-responsive T cells, the

level of proliferation was unexpected and it is unlikely that all the T cell proliferation

reflected in these assays is S. aureus-specific. Certainly some antigens may be conserved

amongst a wide range of bacteria. In addition, there may be some non-specific activation, as

well as death of T cells not receiving activation signals over the five day period which could

diminish the non-proliferative fraction. Enhanced T cell proliferation was further supported

by enhanced IL-2 production in the initial days of the response and a greater than two-fold

increase in IFN-γ by day 5. Although we do not rule out a role for the influence of IL-27 on

DC differentiation having an effect on Th17 cell differentiation and function, the cytokine

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profile suggests a Th1 cell promoting activity of IL-27. Collectively, our data suggests that

IL-27 facilitates a DC phenotype capable of enhanced T cell stimulation. These mechanisms

and the major findings of this report are summarized in Figure 6. In conclusion, our findings

may have important implications for designing DC-based therapeutics and in a variety of

immunological scenarios that concern induction of adaptive immunity such as vaccination.

Acknowledgements

This work was supported by institutional funds supplied by the University of South Carolina

School of Medicine and NIH grant HL093300. Experimental results reported in this

manuscript were collected when the lead and corresponding authors were affiliated with USC.

Subsequent data analysis and manuscript preparation were also performed at Briar Cliff

University and the West Virginia School of Osteopathic Medicine.

Authorship

JYJ and LLR contributed to the design of experiments and were responsible for the

performance of experiments, data analysis and manuscript preparation. CMR and JYJ

conceived the study idea and designed experiments. CMR contributed equally to the

preparation of the manuscript.

Disclosures

The authors declare no conflict of interest.

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Table 1. Monocyte-derived DCs differentiated with IL-27 increase expression of cell surface molecules that facilitate adhesion to and activation of T-cells.

Cell surface molecule

Meda + IL-27a % increase P-Valueb

DC-SIGN 30.53±0.43 45.6±3.93 49.4 <0.05 ICAM-1 81±19.54 107.54±32.82 32.6 >0.05 CD40 31.18±2.66 42.6±4.67 36.6 <0.05 CD11b 322.3±35.39 482.8±71.18 49.8 <0.05 CD18 834.3±98.16 1062±50.02 27.3 <0.05 aHuman monocytes were differentiated to DCs without the addition of IL-27 (Med) or 50 ng/ml of IL-27 (+ IL-27) for 7 days. The mean fluorescent intensity (MFI) is reported ± standard error for four independent blood donors.

bStatistical significance was determined by a Student’s t-test.

Figure legends

Figure 1. DCs were differentiated in the presence or absence of IL-27 as indicated. (A-E)

Cells were subjected to yellow-green fluorescent labeled latex beads for 6 h. (A) Acidified

lysosomes (red) were stained with Lysotracker (200 nM) following latex bead uptake. The

images shown are from an individual experiment representative of three. (B) The MFI

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obtained from untreated or bead-treated DCs differentiated without IL-27 was set to 1.

Results obtained in the presence of IL-27 were expressed relative to this value. The values

presented here are combined results from three independent experiments. (C) DCs were

stained with anti-V-ATPase H antibody as described. Labeled proteins were detected with

anti-mouse IgG conjugated with Alexa-568 (red). Representative images from three

experiments are shown. (D) Percent colocalization was obtained as described in the Methods

section. The values presented here are combined results from three independent experiments.

(E) MFI was obtained as described in the Methods section. The values presented here are

combined results from three independent experiments. (F) Cell lysates were prepared from

DCs differentiated with or without IL-27 for immunoblot analysis of V-ATPase expression.

An image representative of four experiments is shown. (G) The ratio of V-ATPase/actin

band intensity was expressed relative to medium alone for four combined experiments. (B, D,

E, G) A Student’s t test was used to compare ratios from the IL-27-treated group with the

control (MED) at each time point in the 95% confidence interval. (H) Cell lysates were

prepared from DCs differentiated with or without IL-27 for immunoblot analysis of cathepsin

D. Pro; procathepsin D (52kDa), Pre; Precathepsin D (48kDa), mature; mature cathepsin D

(approximately 24 kDa). An image representative of three experiments is shown.

Figure 2. DC differentiation in the presence of IL-27 promotes clearance of Staphylococcus

aureus. DCs were either differentiated with or without IL-27 as indicated. DCs were then

left untreated (Med, IL-27) or pretreated with bafilomycin (100 nM) for four hours, and then

infected with S. aureus for 1 h. Next, gentamycin was added and the DCs were incubated for

an additional 2, 12, or 24 h. Data are represented as the mean CFU recovered from infected

DCs ± SE. Results representative of two independent experiments are shown. Statistical

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significance was determined by a two-way ANOVA and Tukey’s post hoc test for multiple

comparisons. * indicates that the IL-27 group is different from all other treatments; p< 0.05.

Figure 3. HLA-DR expression is enhanced in DCs differentiated in the presence of IL-27.

DCs were differentiated in the presence or absence of IL-27 as indicated. DCs were then

infected with S. aureus for 1 h, treated with gentamycin then incubated an additional 23 h.

(A) Surface labeling of HLA-DR was performed as indicated in the Methods section.

Surface-labeled DCs were analyzed by flow cytometry and representative dot plots are shown.

(B) Quantitative analysis of mean fluorescent intensity (MFI) from of three independent

experiments done with separate blood donors is shown. (C) Quantitative analysis of HLA-DR

gene expression is presented as mean relative change in gene expression of duplicate samples

± SE for two combined experiments. Gene expression was normalized to that of GAPDH and

expressed relative to untreated controls. (B, C)* indicates p<0.001.

Figure 4. DCs differentiated in the presence of IL-27 increased production of IL-12 during S.

aureus infection. DCs were either differentiated with or without IL-27. DCs were then

infected with S. aureus for 1 h, treated with gentamycin and incubated an additional 23 h. (A)

Quantitative analysis of IL-12 p35 and p40 gene expression is presented as mean relative

change in gene expression of duplicate samples ± SE for three combined experiments. Gene

expression was normalized to that of GAPDH and expressed relative to untreated controls. (B)

Supernatants were analyzed for secreted IL-12 p70 by ELISA. Data combining three

independent experiments are shown. * indicates p<0.001.

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Figure 5. DCs differentiated in the presence of IL-27 stimulate enhanced T cell activity.

CFSE-stained T cells are distinguishable from DCs (A-C). Therefore, the CFSE-stained T

cell quadrant (A4) was gated on to exclude DCs from all subsequent analyses. To establish a

T cell proliferative region via CFSE dilution, T cells alone were either left unstimulated (D)

or antibody stimulated (Ab stimulated, E) with plate-bound anti-CD3 and soluble anti-CD28.

Region E (D-G) was then used to enumerate T cell proliferation. DCs differentiated in the

presence (IL-27 DC, G) or absence of IL-27 (Med DC, F), were co-cultured with syngeneic

CD4+ T cells for 5 days. Ten-thousand cells were analyzed for each T cell proliferation assay,

and each assay was performed in duplicate per donor. One representative donor is shown,

and this was repeated with three independent blood donors showing similar results (A-E).

Region E was then used to quantify percent T cell proliferation for three independent blood

donors (H). Statistical significance was determined by a Student’s t-test (H). (I-L)

Supernatants from T cell proliferation assays were analyzed for the production of IL-2 (I, K)

and IFN-γ (J, L). Data from a representative donor time course (I, J), or five combined

donors on day 1 (K) or day 5 (L) are shown. Statistical significance was determined by a

repeated measures paired t test.

Figure 6. Hypothetical model. Exposure to IL-27 during DC differentiation leads to a

number of functional changes. Increased lysosomal acidification is mediated by a greater

expression of V-ATPase and promotes improved antigen processing. MHC class II

expression is increased allowing for enhanced presentation of antigen to T cells. DC-SIGN,

CD40, and MAC-1 surface expression is increased strengthening the interaction with T cells.

These changes combined with augmented IL-12 production enhance T cell proliferation and

effector cytokine production.

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the presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes...

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