Interleukin-27 and interleukin-12 augment activation of distinct cord blood natural killer cells responses via STAT3 pathways

Journal of the Formosan Medical Association (2012) 111, 275e283

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journal homepage: www.jfma-online.com

ORIGINAL ARTICLE

Interleukin-27 and interleukin-12 augment activation of distinct cord blood natural killer cells responses via STAT3 pathways Juei-Chang Chen a,y, Ai-Ju Huang b,y, Shih-Chang Chen c, Jia-Long Wu b, Wen-Mein Wu d, Han-Sun Chiang b, Chia-Hao Chan b, Chih-Ming Lin e, Yu-Tzu Huang b,* a

Department of Medical Research, Tao-Yuan General Hospital, Taoyuan, Taiwan School of Medicine, Fu Jen Catholic University, 510 Chung-Cheng Road, New Taipei City, Taiwan c Chen’s Woman Clinical Hospital, New Taipei City, Taiwan d Department of Nutritional Science, Fu Jen Catholic University, 510 Chung-Cheng Road, New Taipei City, Taiwan e Department of Surgery, Cathay General Hospital, Taipei, Taiwan b

Received 8 October 2010; received in revised form 21 October 2010; accepted 22 October 2010

KEYWORDS cord blood; interleukin-27; interleukin-12; glycoprotein-130; natural killer cells; STAT3

Background/Purpose: Umbilical cord blood is rich in primitive natural killer (NK) cells, which are activated by interleukin (IL)-12. It was previously reported that a novel IL-12 family cytokine, IL-27 comprised of EBI3 and p28, was elevated in maternal serum during normal pregnancy. Thus, we compared the immune regulatory functions of IL-27 and IL-12 on mononuclear cells derived from cord blood and adult peripheral blood. Methods: After stimulation with IL-27, IL-12, and IL-27 combined with IL-12, the cytotoxicity against BJAB lymphoma cells by blood mononuclear cells was performed. Then immunofluorescence staining, reverse transcriptase-polymerase chain reaction and Western blotting were used to detect the effects of IL-27 and IL-12 in isolated NK cells. Results: IL-27, IL-12, and IL-27 combined with IL-12 enhanced the cytotoxicity of adult peripheral blood cells and cord blood cells, but the proliferation of distinct subpopulations of cells was not evident. Similar results were also obtained with purified cord blood NK cells. Interestingly, distinct from IL-12, IL-27 could induce aggregation and morphological changes of umbilical cord blood cells. Finally, IL-27 combined with IL-12 could stimulate increased IL-27 receptor (gp130 and WSX-1) transcripts in purified cord blood NK cells. However, the activation of signal transducer and activator of transcription 3 (STAT3) in NK cells was only detected in the presence of IL-27, but not IL-12 alone.

* Corresponding author. School of Medicine, Fu Jen Catholic University, 510 Chung-Cheng Road, New Taipei City 24205, Taiwan. E-mail address: [email protected] (Y.-T. Huang). y J.-C. Chen and A.-J. Huang contributed equally to this work. 0929-6646/$ - see front matter Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved. doi:10.1016/j.jfma.2010.10.002

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J.-C. Chen et al. Conclusion: From previous results, we summarize our current understanding of the augmentation of distinct regulation of NK cells by IL-27 and IL-12. Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.

Introduction Numerous tumor cells or microbe-infected cells can escape the cytotoxicity of CD8þ T-cells by downregulating major histocompatability complex (MHC) class I complex and accessory molecules. However, these deficient cells may be eliminated as the targets of natural killer (NK) cells. NK cells are one of the cellular mediators of innate immune responses that do not require adaptive immunization. During developmental progression, NK cells express various cell surface molecules and cytokine receptors, which are markers that can be used to identify the stages of NK cell development.1 In addition, upon cytokine stimulation and transcription factor activation, NK cells can progress to mature stages, at which time they can produce interferongamma and enhance cytotoxicity via the production of perforins and granzymes. The cytotoxic capability of NK cells is regulated by the balance of signals mediated by inhibitory and activating receptors of NK cells.2,3 On the surfaces of target cells, self-MHC class I molecule expression is a crucial signal for inhibition, whereas ligands for the very large ITAM-bearing or DAP10-associated transducing receptors are involved with activation.4 In addition, the signals arising from cytokine receptors, when integrated with paracrine interleukin (IL)-12, IL-15, tumor necrosis factor-alpha, and interferons, are also involved in the maturation and activation of NK cells.5 IL-12 enhances the responses of T-helper type 1 (Th1) cells and reactivation of memory CD4þ T-cells.6e8 Additional evidence supports its critical roles for priming the cytotoxicity of CD8þ T-cells and NK cells by promoting the expression of cytolytic factors, such as perforin and granzymes.9,10 IL-27, a novel member of the IL-12 family, is composed of the EpsteineBarr viral-induced gene 3 (EBI3) and p28. EBI3 is an IL-12 p40-related protein, and p28 is a newly discovered IL-12 p35-related protein.11 In recent studies, IL-27 also had the ability to enhance the trend toward Th1 immune responses by inducing the Th1-specific transcription factor T-bet.12 In addition, IL-27 was shown to have potent antitumor activity as it acted directly on CD8þ T-cells and enhanced granzyme B expression.13 The biological functions of IL-12 are dependent on the IL-12 receptor subunits b1 and b2 expressed on CD4þ T-cells, CD8þ T-cells, and NK cells.14,15 Recently, receptors for IL-27 were defined. These included a homolog of the IL-12 receptor b2 subunit, WSX-1 (T-cell cytokine receptor), and a functional signal transducing receptor, gp130.11,16 Compared with IL-12 receptors, the presence of WSX-1/ gp130 receptors indicates that IL-27 has other biological functions distinguishable from those of IL-12. WSX-1 can be constitutively expressed by a range of lymphoid cells, including NK, NKT, CD4þ T, and CD8þ T-cells.17 gp130 is widely expressed in various organs. It is a common signal transducer of the IL-6 family, including IL-6, leukemia inhibitory factor, IL-11, ciliary neurotrophic

factor, oncostatin M, cardiotropin-1, and stimulating neurotrophin-1/B-cell stimulating factor-3.18 Activation of gp130 can promote cell proliferation and survival, leading to the inhibition of an apoptotic signaling pathway.19,20 gp130, as an initial cell signal transducer, leads to activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in naive CD4þ T-cells and enhances their proliferation.21,22 Recently, studies have indicated that IL-27 enhances lymphocyte differentiation via two distinct independent pathways: p38 MAPK/ T-bet and ICAM-1/LFA-1/ERK1/2.23,24 Although the functions of IL-27 in the development and activation of human T-cells have been reasonably well defined, information regarding the signals from IL-27 to regulate human NK cell cytotoxicity is very limited. Therefore, additional definitions of the functions of IL-27 in NK cells would be valuable for understanding its mechanisms related to innate immunity. In this study, expression systems for IL-27 and IL-12 were established in vitro. We demonstrate that IL-27 and IL-12 augment the activation of distinct cytotoxicity responses of BMCs from adult peripheral blood and cord blood. In addition, enhanced cytotoxic activity was confirmed using isolated cord blood NK cells. Further, activation of several transduction mediators, including the IL-27 receptor (gp130 and WSX-1) and STAT3, were defined in the IL-27- and IL-12-responsive cells.

Materials and methods Expressions of IL-27, IL-12, and EBI3 Expression plasmids for IL-27 (EBI3-FLAG/pRK5F and p28FLAG/pRK5F) and IL-12 (p40-FLAG/pRK5F and p35-FLAG/ pRK5F) were generated based on a pRK5F vector that contained a CMV promoter/enhancer and FLAG terminus. The gene for EBI3 was amplified from a human placenta complementary DNA (cDNA) library. The p28 gene was derived from an embryonic stem cell clone. The genes for the two subunits of IL-12, p40 and p35, were derived from phytohemagglutinin-stimulated human peripheral BMCs. The open reading frames of EBI3, p28, p40, and p35 were amplified using polymerase chain reaction (PCR) technology and then inserted into pRK5F vectors. The expression plasmids for IL-27, IL-12, IL-27þIL-12, and EBI3 were transfected into 293 T cells using the method of CaCl2 transfection. The supernatants derived from transient 293 T transfectants were analyzed by Western blotting using anti-FLAG M2 monoclonal antibodies, and then expressions of the IL-27 (EBI3 and p28) and IL-12 (p40 and p35) heterodimers were detected. In addition, when IL-27- and IL-12-specific enzyme-linked immunosorbent assay (ELISA; eBioscience, California, CA, USA) was used, the IL-27 and IL-12 proteins expressed in all 293 T transfectants were found to be

Effect of IL-27 on cord blood natural killer cells appreciable and identical. Finally, similar concentrations of IL-27 (150 ng/mL), IL-12 (150 ng/mL), and IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12) were used for stimulating BMCs and NK cells.

Isolation of BMCs from cord blood and adult peripheral blood

277 cells with green fluorescence membrane (FL1) and red fluorescence nuclei (FL3) staining were analyzed by flow cytometry. BJAB target cells killed by NK cells were then identified by double-fluorescent staining (a double positive region of FL1 and FL3). The proportion of killed cells was determined by comparison with total target cell numbers.

Flow cytometry All human blood samples, either adult peripheral blood or cord blood, were obtained with the approval of the Institutional Review Board of Fu Jen Catholic University (IRB No. C9717). Blood was centrifuged at 250 g for 20 minutes at room temperature to separate the cells from the plasma. The plasma was removed, blood cells were mixed with an equal volume of RPMI 1640 medium containing 10% fetal bovine serum (FBS), and these were then layered onto Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). After centrifuging at 250 g for 30 minutes, the buffy coat at the interphase was collected. The isolated BMCs were resuspended in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin, and were used for cytotoxicity assays.

Cell suspensions obtained from peripheral BMCs, cord blood BMCs, or purified NK cells were pelleted and resuspended in staining medium (RPMI 1640 containing 1% FBS and 0.1% sodium azide). Various fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies, directed against CD4, CD8, CD16, and CD56 (all from BioLegend, San Diego, California, USA), were added to the staining medium. After 1 hour’s incubation at 4  C, unbound monoclonal antibody was removed by washing the cells with washing medium (phosphate-buffered saline plus 1% FBS and 0.1% sodium azide). Cell pellets were resuspended in staining buffer and analyzed by immunoflow cytometry on a FACScan (Becton Dickinson Biosciences, San Jose, CA, USA) using BD CellQuest software.

Isolation of NK cells Reverse transcription PCR Blood cells were incubated with CD56e and CD16e selection paramagnetic beads and then layered onto Ficoll-Hypaque. NK cells were negatively selected from cells using markers for CD3, CD4, CD19, CD36, and CD66b from a Human NK Cell Enrichment Cocktail (Rosette Sep. StemCell Technologies, Inc., Vancouver, BC, Canada). After centrifuging at 1000 g for 30 minutes, the buffy coat at the interphase was collected and the cells were washed once with RPMI 1640 medium. The washed isolated NK cells were resuspended in medium supplemented with 10% FBS and were used for cytotoxicity assays. Isolated NK cells were confirmed by immunoflow cytometry, with a purity of up to 86%. These isolated NK cells were used for cytotoxicity assays, and their total protein was extracted and prepared for immunoblotting.

Cell culture NK cells, BMCs, and BJAB lymphoma cells were cultured in RPMI 1640 medium containing 10% FBS, 2 mM glutamine, 50 units/mL penicillin, and 50 mg/mL streptomycin. 293 T cells were incubated in Dulbecco’s Modified Eagle Medium-based medium with 10% FBS, 2 mM glutamine, 50 U/mL penicillin, and 50 mg/mL streptomycin.

Cytotoxicity assay According to the manufacturer’s instructions, the standard assays of LIVE/DEAD Cell-Mediated Cytotoxicity (Molecular Probes, Inc, Eugene, OR, USA) were modified to measure NK cell-mediated cytotoxicity by fluorescence flow cytometry assays. The target BJAB cells were labeled with green fluorescent DiOC18 (FL1), and then combined in various proportions with effector cells. After cellecell contact for 4 hours at 37 C, the nucleic acid from all the cells was counterstained with propidium iodide (PI). Dead target

Total RNA was extracted from cells using REzol C&T reagent (Protech Technology Co, Taipei, Taiwan) according to the manufacturer’s instructions. Single-stranded cDNA was then generated in a 20 mL reverse transcription (RT) reaction volume containing 1 mg RNA as template, random primers, and SuperScript II RNase He reverse transcriptase (Promega Co, Madison WI, USA). RT products (1 mL) were used for PCR amplification in a 50 mL reaction volume containing sense and antisense primers for gp130 and WSX-1 (400 nM), dATP, dGTP, dTTP, and dCTP mixture (each 200 mM), 1  PCR reaction buffer, and DNA polymerase (Protech Technology Co). PCR products were electrophoresed on 1.6% agarose gels and visualized with ethidium bromide staining under ultraviolet light. Simultaneously, transcripts encoding glyceraldehyde 3-phosphate dehydrogenase were monitored in all samples and served as an internal control. The primers used are shown in Table 1. Table 1 Nucleotide sequences of primers for the reverse transcription polymerase chain reaction. Target genes (size of products)

Nucleotide sequence

gp130 (168 bp) Forward primer Reverse primer

50 -ATACCTTAAACAAGCTCCACCTTC-30 50 -AGTTTCATTTCCAATGATGGTTCT-30

WSX-1 (183 bp) Forward primer Reverse primer

50 -GAAACCCAAATGAAGCCAAA-30 50 -GCCTCCTGACATCTTCGGTA-30

GAPDH (108 bp) Forward primer Reverse primer

50 -TTGGTATCGTGGAAGGACTC-30 50 -CAGTAGAGGCAGGGATGAT-30

GAPDH Z glyceraldehyde 3-phosphate dehydrogenase.

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Western blot assays Culture supernatants (10 mL) collected from 293 T cell transfectants were incubated with lysis buffer [0.6% NP-40, 154 mM NaCl, 10 mM Tris-HCl (pH7.4), 1 mM EDTA, and 0.1% sodium dodecyl sulfate (SDS)] for 5 minutes, and then the expressions of IL-27 and IL-12 were examined. To detect STAT3 and phospho-STAT3, IL-27- or IL-12-stimulated BMCs or purified NK cells were washed with phosphate-buffered saline, and then incubated with lysis buffer for 5 minutes. Cells were sonicated on ice for 2 minutes, and cell lysates were cleared by centrifugation at 14,000 g for 10 minutes. Culture supernatants or cell lysates were resolved by 10% SDS-polyacrylamide gel electrophoresis (Bionovas Biotechnology Co., Toronto, ON, Canada). The isolated proteins were transferred to a nitrocellulose membrane. The membrane was reacted with primary monoclonal antibodies recognizing FLAG peptide (M2 monoclonal antibodies; Sigma, Inc, Saint Louis, MO, USA), STAT3 (Cell Signaling Technology, Inc, Danvers, MA, USA), or phospho-STAT3 (Cell Signaling Technology, Inc), followed by horseradish peroxidase-conjugated secondary antibody (Millipore, Inc, Billerica, MA, USA). The blot was developed using enhanced chemiluminescence.

Statistical analysis Results are given as means  standard errors of the mean. Statistical comparisons between two groups used the Student t test. Analysis of variance followed by Scheffe’s test was used for multiple group comparisons. A p value of <0.05 was considered to be statistically significant.

Results IL-27 and IL-12 enhance the cytotoxic activities of BMCs derived from adult peripheral blood and cord blood It is well known that IL-12 enhances the cytotoxicity of NK cells.25 In addition, the two subunits of the IL-27 heterodimer are similar to those of IL-12. In an attempt to define functional characteristics of IL-27 and IL-12, the cytotoxic activity of NK cells was employed based on assay systems of cell-mediated cytotoxicity (Fig. 1). As described above,

Figure 1 Stimulation effects of IL-27 and IL-12 on cytotoxic activity of BMCs derived from adults or cord blood. (A) BMCs collected from cord blood were stimulated with IL-27 (150 ng/ mL), IL-12 (150 ng/mL), or IL-27þIL-12 (containing 150 ng/mL IL-27 and approximate 150 ng/mL IL-12) for 24 hrs, and then

used as effector cells. DiOC18-labeled BJAB lymphoma cells were as the target cells. Activated BMCs were co-cultured with target cells at an effector cell to target cell ratio of 50:1 for 4 hrs, and then total cells were stained with PI. Inset shows flow cytometric profiles of an aliquot of the target cells stained with DiOC18 (FL1) and PI (FL3). The percentages of dead target cells were determined by double-positive fluorescence cells/total FL1 positive cells in the cytotoxic activity assays. BMCs collected from (B) cord blood or (C) adult peripheral blood were stimulated with IL-27, IL-12, IL-27þIL-12, or EBI3 for 24 hrs, and the cytotoxic activity assays were performed. Results (mean  SEM) are representative of five independent experiments. (*p < 0.05 vs. vector control at an effector cell to target cell ratio of 25:1, #p < 0.05 vs. vector control at an effector cell to target cell ratio of 50:1).

Effect of IL-27 on cord blood natural killer cells isolated cord blood BMC effector cells were co-cultured with DiOC18-labeled BJAB lymphoma target cells in different proportions (BMC to BJAB cell ratios of 50:1). After cellecell contact for 4 hours, nucleic acid was counterstained with PI. It should be noted that, distinct from antigen-specific cytotoxic T-cells, NK cells only require short periods of antigen stimulation and kill target cells within several hours. The dead BJAB target cells had concomitant staining with DiOC18 in the cell membrane and PI staining of the nuclei; these were analyzed by flow cytometry. Thus, target cells killed by NK cells had double fluorescent labels (a double positive region of FL1 and FL3). The proportion of cells killed due to cytotoxicity was determined by the percentage of double-positive cells with reference to the total number of DiOC18-labeled target cells. The BMCs from adult peripheral blood and cord blood were analyzed. After stimulation with IL-27 (150 ng/mL), IL-12 (150 ng/mL), IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12), and EBI3 (150 ng/mL) for 24 hours, the percentages of lysed BJAB target cells were determined. These results showed that IL-27 and IL-12 could enhance the cytotoxic activities of BMCs derived from cord blood (Fig. 1) and adult peripheral blood (Fig. 1). In addition, IL-27 combined with IL-12 significantly promoted this killing activity. Interestingly, the cytotoxic activity of BMCs derived from cord blood was higher than that of BMCs derived from adult peripheral blood. IL-27þIL-12 significantly stimulated cord blood BMCs at an effector to target cell ratio of 1:50. However, EBI3 alone, one subunit of

279 IL-27, did not induce cytotoxicity of BMCs derived from either peripheral blood or cord blood.

IL-27 stimulates aggregation of BMCs In order to examine the activation characteristics of IL-27 compared with those of IL-12 on BMCs, we observed that the morphologies of cord blood BMCs changed when they were cultured in medium containing IL-27 (150 ng/mL), IL-12 (150 ng/mL), and IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12) for 48 hours. After removing the culture supernatants and suspended cells, a considerable number of BMCs were found to be adherent to the culture dishes when stimulated with IL-27 and IL-27þIL-12 (Fig. 2). In addition, IL-27 and IL-27þIL-12 stimulated BMCs to form aggregates. These aggregated BMCs were shown to contain 41% CD4þ, 22% CD8þ, and 10% NK cells using specific antibodies against cell surface markers and immunofluorescence staining (data not shown). These results indicated that Th cells, cytotoxic T (Tc) cells, and NK cells were stimulated in the presence of IL-27 and IL-27þIL-12.

Activation of cytotoxicity does not result from proliferating BMC subpopulations In order to exclude cell proliferation from IL-27-enhanced stimulation, we determined the numbers and percentages

Figure 2 Observation of aggregated cord blood cells after stimulation with cytokines. Cord blood mononuclear cells (BMCs) were isolated and cultured in medium containing interleukin (IL)-27 (150 ng/mL), IL-12 (150 ng/mL), and IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12). After 48 hours, cells in suspension were removed and the distributions and morphologies of cord BMCs were observed on the culture dish using a light microscope.

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of cell subpopulations among BMCs stimulated with cytokines. After stimulation with IL-27, IL-12, and IL-27þIL-12 for 48 hours, cell subpopulations of BMCs derived from adult peripheral blood cord blood were determined as described above. As shown in Fig. 3, among adult peripheral and cord blood BMCs, Th cells (CD4þ), Tc cells (CD8þ), and NK cells (CD16þCD56e and CD16þCD56þ) were significantly detected. As shown in Fig. 3, there was no change in the absolute number of cells after stimulation with IL-27, IL-12, and IL-27þIL-12 for 2 days. This indicated that IL-27 or IL-12 would not induce cellular proliferation during a short period of time. However, compared with the vector control, the percentages of Th, Tc, and NK cells were similar after stimulation with either IL-27 or IL-12. Therefore, the

% of cell subpopulations

A

60 Vector

50

IL-27 IL-12

40

IL-27+IL-12

30 20 10 0 Th cells

% of cell subpopulations

B

Tc cells

NK cells

60 50

Vector IL-27

40

IL-12 IL-27+IL-12

absolute numbers of Th, Tc, and NK cells in cord blood were consistent and identical after treatment with cytokines.

IL-27 stimulates the cytotoxic activity of purified NK cells derived from cord blood Cord blood BMCs were stimulated with IL-27 (150 ng/mL), IL-12 (150 ng/mL), and IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12) for 18 hours. NK cells were then purified by negative selection methods. The isolated NK cells were confirmed by immunoflow cytometry, with a purity of up to 86%. These purified NK cells were subsequently co-cultured with BJAB target cells for 4 hours. The cytotoxic capability of these purified NK cells was determined as described above. As shown in Fig. 4, compared with a vector control, cell-mediatedcytotoxicity was enhanced after stimulation with IL-27, IL-12, or IL-27þIL-12.

IL-27 induces the expression of IL-27 receptors and the phosphorylation of STAT3 in cord blood NK cells It has been shown that IL-27 activates T-cells via IL-27 receptors (gp130 and WSX-1). Therefore, we sought to confirm whether IL-27 activation of NK cells could also occur through similar pathways. NK cells purified from cord blood BMCs were stimulated with IL-27 for 36 hours, and then IL-27 receptor transcripts (gp130 and WSX-1) were detected by RT-PCR. Fig. 5A shows that WSX-1 was constitutively expressed by NK cells. Interestingly, IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12) was able to induce the expression of gp130. This indicated that IL-27 receptors could be detected in the NK cells of cord blood, especially in the presence of IL-12þ IL-27. In addition, activation signaling was confirmed in

30 20 10 0 Th cells

Tc cells

NK cells

Figure 3 Percentages of cell subpopulations of blood mononuclear cells (BMCs) derived from adult peripheral blood or cord blood after stimulation with interleukin (IL)-27/IL-12. Cells in suspension for BMCs from adult peripheral blood (A) or cord blood (B) were stimulated with IL-27 (150 ng/mL), IL-12 (150 ng/mL), and IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12) for 2 days. Stimulated BMCs were stained with fluorescent-labeled monoclonal antibodies against CD3 and CD4 [markers of T helper lymphocytes (Th cells)], CD3 and CD8 [markers of cytotoxic T lymphocytes (Tc cells)] or CD56 and CD16 (markers of NK cells). The phenotypes of the cells in the stimulated BMCs were analyzed using an immunoflow cytometric assay.

Figure 4 Cytotoxic activity of interleukin (IL)-27/IL-12stimulated purified natural killer (NK) cells from cord blood. Fresh paramagnetic bead-selected NK cells were isolated from cord blood and then stimulated with vector control, IL-27, IL12, IL-27 þ IL-12, or EBI3 for 24 hours. The percentages of BJAB cells killed by cytokine-pretreated NK cells were determined for an NK cell to target cell ratio of 2:1. The results (mean  standard errors of the mean) were representative of three independent experiments. *p < 0.05 versus vector control.

Effect of IL-27 on cord blood natural killer cells

281 stimulated NK cells. After adding IL-27 (150 ng/mL), IL-12 (150 ng/mL), or IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12) to purified cord blood NK cells for 4 hours, total cellular protein was isolated. It is known that gp130 can induce the activation of STATs; therefore, STAT3 and phospho-STAT3 expression by NK cells was analyzed by Western blotting. The results, shown in Fig. 5B, indicate that IL-27, as well as IL-27þIL-12, enhanced the expression of phospho-STAT3 in cord blood NK cells. However, IL-12 had no effects on stimulating STAT3. These results indicated that IL-27 activated cord blood NK cells via gp130 and STAT3 pathways.

Discussion

Figure 5 Regulation of interleukin (IL)-27/IL-12 in freshly isolated cord blood cells. (A) Expression of gp130 in isolated cord blood natural killer (NK) cells. After stimulation with or without IL-27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12) for 24 hours, purified NK cells were collected. Cord blood NK cells were then isolated using microbeads, and the transcripts for IL-27 receptors (gp130 and WSX-1) were detected by the reverse transcription polymerase chain reaction. The transcripts of phytohemagglutininstimulated adult peripheral blood mononuclear cells (PBMCs) were used as a positive control. (B) IL-27 and IL-12 signaling in isolated cord blood NK cells. NK cells from cord blood were stimulated with IL-27 (150 ng/mL), IL-12 (150 ng/mL), IL27þIL-12 (containing 150 ng/mL IL-27 and approximately 150 ng/mL IL-12), or EBI3 (150 ng/mL). After 4 hours, total protein was isolated from stimulated NK cells, and STAT3 activation signals were detected by phospho-STAT3 (p-STAT3) and STAT3 immunoblotting. Phosphorylated STAT3 levels were normalized to total protein levels, and were presented as band densities. The results (mean  standard error of the mean) were representative of at least three independent experiments. *p < 0.05 versus vector control. GAPDH Z glyceraldehyde 3-phosphate dehydrogenase.

In this study, we have defined immunomodulatory activities of IL-27 and IL-12 when acting on cord blood cells and adult peripheral BMCs. We first constructed plasmids containing genes for EBI3, p28, p40, and p35, and established expression systems for IL-27 and IL-12 in transfectants. Cytotoxicity assays for NK cells were also established in vitro. Our results showed that IL-27, IL-12, and IL-27þ IL-12 could enhance the cytotoxicity capability of adult peripheral BMCs (Fig. 1). Moreover, as shown in Fig. 1, IL-27, IL-12, and IL-27þIL-12 significantly augmented the cytotoxicity and activity of umbilical cord blood cells. This revealed that umbilical cord blood cells were more sensitive to stimulation by IL-27/IL-12. However, these results do not rule out the possibility that other subpopulations of umbilical cord blood cells contribute to cellular cytolysis. Therefore, we isolated cord blood NK cells and determined the killing capability of these cells in the presence of IL-27 and IL-12. Interestingly, IL-27/IL-12 did indeed enhance the activities of cord blood NK cells (Fig. 4). It is known that IL-27 is elevated in the placenta and maternal serum during normal pregnancy.26,27 We also investigated whether IL-27 receptors (gp130 and WSX-1) were detectable in umbilical cord blood NK cells, especially after stimulation with IL-27 and IL-12. Our results indicated that gp130 protein may be inducible, and that IL-27 takes a period of time (24e48 hours) for induction of its activation (Fig. 5A). In addition, experiments to define the signals underlying the immune enhancement effects of IL-27 were carried out, as shown in Fig. 5B. IL-12 family members include IL-27, IL-23, and IL-35, based on their subunit combinations and receptor structures. It has previously been shown that each IL-12 family member contributes to the differentiation of Th cells with different roles. After stimulation with 150 ng/mL of IL-27 or IL-12, IL-27 showed an ability to stimulate BMC clumping, compared with IL-12. This indicated that IL-27 might induce more adhesion molecules, such as ICAM-1/LFA-1, or CCR5dan interesting topic for further studies. IL-12 receptors b1 and b2 triggered the activation of JAK-STAT signaling pathways during Th1 differentiation, and STAT1 and STAT4 were the predominant mediators. In addition, IL-23 triggered the development of memory Th1 cells and the differentiation of Th17 cells via the activation of STAT3 and STAT4.14 IL-27 is a key cytokine to drive Th0 cells to become Th1 cells during the initiation of Th1 cell differentiation via STAT1 and STAT3. However, during IL-27

282 stimulation, phosphorylation of STAT3 was detected in the isolated cord blood NK cells. This result has also been observed with murine DX-5þ NK cells.24 By comparison with IL-27, stimulation with IL-12 did not enhance the activation of STAT3 in cord blood NK cells. In conjunction with our results, this indicates that IL-27 and IL-12 affect the cytotoxicity of NK cells through different receptor/signal pathways. Recently, cytokine inhibitory proteins, suppressors of cytokine signaling (SOCS), were discovered, and their functions associated with the IL-12/IL-6 family were defined.28,29 IL-12-induced activation of T-cell proliferation and NK cell cytotoxicity were enhanced in SOCS-deficient cells. Further, in the absence of SOCS expression, IL-27 was able to enhance the proliferation of murine T-cells. In our study, cord blood BMCs showed greater activation than adult peripheral BMCs in response to IL-27. Therefore, after stimulation with IL-27, the expressions and contributions of SOCS proteins in cord blood BMCs warrant investigation. NK cells are derived from adult human hematopoietic stem cells in the bone marrow. During developmental progression, NK cells can express a variety of cell surface molecules and cytokine receptors, markers that can be used to identify the stages of NK cell development.30e34 In addition, using cytokine stimulation and transcription factor activation, NK cells progress to mature stages, at which time they can become cytotoxic and produce interferon-gamma. Studies of mice or humans with gene deficiencies for cytokine receptors or nuclear factors have shown that the microenvironment could affect the development and differentiation of NK cells. It has been shown that umbilical cord blood is a possible alternative source for allogeneic stem cell transplantation.35 However, umbilical cord blood is rich in primitive NK cells, which are expanded by host cytokines and posses cytotoxic capacities. This could give rise to substantial graft reactions when used for treating leukemia.36,37 Therefore, based on our results, we would anticipate that the application of IL-27, with characteristics of cytotoxicity regulation, could improve the side effects associated with allogeneic stem cell transplant therapy. The enhanced responses of cord blood NK cells to IL-27 may also contribute to neonatal immune defense and antitumor effects during cord blood stem cell transplantation.38,39

Authorship J.-C. C., A.-J. H., C.-M. L., and Y.-T. H. designed the study and analyzed the data. A.-J. H., S.-C. C., C.-L. W., and Y.-T. H. performed experiments and wrote the manuscript. A.-J. H. and C.-H. C. performed experiments and analyzed the data. W.-M. W., C.-L. W., and H.-S. C. took part in the experimental design and analyzed the data. Funding for the project was acquired by J.-C. C, C.-M. L., and Y.-T. H. All authors contributed to the writing of the manuscript.

Acknowledgment This work was supported by National Science Council grant NSC 96-2320-B-030-002-MY2, collaboration from Tao-Yuan

J.-C. Chen et al. General Hospital and Fu Jen Catholic University grant TYGHSTU-95B-1, and collaboration of Cathay General Hospital and Fu Jen Catholic University grant CGH-FJU-97-08.

References 1. Huang YT, Chen CL, Chou SH, Chiang HS, Chen JC. Characteristics of hematopoietic stem cells developing into natural killer cells and their clinical application. Fu-Jen J Med 2006;4:s41e9. 2. Bottino C, Castriconi R, Moretta L, Moretta A. Cellular ligands of activating NK receptors. Trends Immunol 2005;26:221e6. 3. Young LS, Rickinson AB. Epstein-barr virus: 40 years on. Nat Rev Cancer 2004;4:757e68. 4. Vivier E. What is natural in natural killer cells? Immunol Lett 2006;107:1e7. 5. Wu J, Lanier LL. Natural killer cells and cancer. Adv Cancer Res 2003;90:127e56. 6. Curtsinger JM, Lins DC, Mescher MF. Signal 3 determines tolerance versus full activation of naive cd8 t cells: dissociating proliferation and development of effector function. J Exp Med 2003;197:1141e51. 7. Kalinski P, Hilkens CMU, Wierenga EA, Kapsenberg ML. T-cell priming by type-1and type-2 polarized dendritic cells: the concept of a third signal. Immunol Today 1999;20:561e7. 8. Jae KY, Jae HC, Seung WL, Young CS. IL-12 provides proliferation and survival signals to murine CD4 þ T cells through phosphatidylinositol 3-kinase/akt signaling pathway. J Immunol 2002;169:3637e43. 9. DelVecchio M, Bajetta E, Canova S, Lotze MT, AmyWesa, Parmiani G, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res 2007;13:4677e85. 10. Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3: 133e46. 11. Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, et al. IL-27, a heterodimeric cytokine composed of ebi3 and p28 protein, induces proliferation of naive CD4(þ) T cells. Immunity 2002;16:779e90. 12. Kamiya S, Owaki T, Morishima N, Fukai F, Mizuguchi J, Yoshimoto T. An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4 þ T cells. J Immunol 2004;173:3871e7. 13. Morishima N, Owaki T, Asakawa M, Kamiya S, Mizuguchi J, Yoshimoto T. Augmentation of effector CD8 þ T cell generation with enhanced granzyme B expression by IL-27. J Immunol 2005;175:1686e93. 14. Trinchieri G, Pflanz S, Kastelein RA. The IL-12 family of heterodimeric cytokines: new players in the regulation of t cell responses. Immunity 2003;19:641e4. 15. Gee K, Guzzo C, Mat NFC, Ma W, Kumar A. The IL-12 family of cytokines in infection, inflammation and autoimmune disorders. Inflamm Allergy Drug Targets 2009;8:40e52. 16. Pflanz S, Hibbert L, Mattson J, Rosales R, Vaisberg E, Bazan JF, et al. Wsx-1 and glycoprotein 130 constitute a signaltransducing receptor for IL-27. J Immunol 2004;172:2225e31. 17. Villarino AV, Larkin 3rd J, Saris CJ, Caton AJ, Lucas S, Wong T, et al. Positive and negative regulation of the IL-27 receptor during lymphoid cell activation. J Immunol 2005;174:7684e91. 18. Kishimoto T, Taga T, Akira S. Cytokine signal transduction. Cell 1994;76:253e62. 19. Yamauchi-Takihara K, Kishimoto T. Cytokines and their receptors in cardiovascular diseases e role of gp130 signalling pathway in cardiac myocyte growth and maintenance. Int J Exp Pathol 2000;81:1e16. 20. Hirota H, Chen J, Betz UA, Rajewsky K, Gu Y, Ross Jr J, et al. Loss of a gp130 cardiac muscle cell survival pathway is

Effect of IL-27 on cord blood natural killer cells

21. 22.

23.

24.

25.

26.

27.

28.

a critical event in the onset of heart failure during biomechanical stress. Cell 1999;97:189e98. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood 1995;86:1243e54. Hirano T, Ishihara K, Hibi M. Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors. Oncogene 2000; 19:2548e56. Owaki T, Asakawa M, Fukai F, Mizuguchi J, Yoshimoto T. IL-27 induces Th1 differentiation via p38 MAPK/T-bet- and intercellular adhesion molecule-1/LFA-1/ERK1/2-dependent pathways. J Immunol 2006;177:7579e87. Matsui M, Kishida T, Nakano H, Yoshimoto K, Shin-Ya M, Shimada T, et al. Interleukin-27 activates natural killer cells and suppresses NK-resistant head and neck squamous cell carcinoma through inducing antibody-dependent cellular cytotoxicity. Cancer Res 2009;69:2523e30. Ogata KTH, Yokose N, An E, Dan K, Hamaguchi H, Sakamaki H, et al. Effects of interleukin-12 on natural killer cell cytotoxicity and the production of interferon-gamma and tumour necrosis factor-alpha in patients with myelodysplastic syndromes. Br J Haematol 1995;90:15e21. Coulomb-L’Hermine A, Larousserie F, Pflanz S, Bardel E, Kastelein RA, Devergne O. Expression of interleukin-27 by human trophoblast cells. Placenta 2007;28:1133e40. Mas AE, Petitbarat M, Dubanchet S, Fay S, Lede ´e N, Chaouat G. Immune regulation at the interface during early steps of murine implantation: involvement of two new cytokines of the IL-12 family (IL-23 and IL-27) and of TWEAK. Am J Reprod Immunol 2008;59:323e38. Eyles JL, Metcalf D, Grusby MJ, Hilton DJ, Starr R. Negative regulation of interleukin-12 signaling by suppressor of cytokine signaling-1. J Biol Chem 2002;277:43735e40.

283 29. Brender C, Tannahill GM, Jenkins BJ, Fletcher J, Columbus R, Saris CJM, et al. Suppressor of cytokine signaling 3 regulates CD8 T-cell proliferation by inhibition of interleukins 6 and 27. Blood 2007;110:2528e36. 30. Miller JS, Alley KA, McGlave P. Differentiation of natural killer (NK) cells from human primitive marrow progenitors in a stroma-based long-term culture system: identification of a CD34 þ 7 þ NK progenitor. Blood 1994;83:2594e601. 31. Blom B, Spits H. Development of human lymphoid cells. Annu Rev Immunol 2006;24:287e320. 32. Farag SS, Caligiuri MA. Human natural killer cell development and biology. Blood Rev 2006;20:123e37. 33. Vosshenrich CA, Samson-Villeger SI, Di Santo JP. Distinguishing features of developing natural killer cells. Curr Opin Immunol 2005;17:151e8. 34. Di Santo JP. Natural killer cell developmental pathways: a question of balance. Annu Rev Immunol 2006;24:257e86. 35. Farag SS, Fehniger TA, Ruggeri L, Velardi A, Caligiuri MA. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 2002;100:1935e47. 36. Cooper MA, Fehniger TA, Turner SC, Chen KS, Ghaheri BA, Ghayur T, et al. Human natural killer cells: a unique innate immunoregulatory role for the CD56bright subset. Blood 2001; 97:3146e51. 37. Hao Q-L, Zhu J, Price MA, Payne KJ, Barsky LW, Crooks GM. Identification of a novel, human multilymphoid progenitor in cord blood. Blood 2001;97:3683e90. 38. Haddad R, Guardiola P, Izac B, Thibault C, Radich J, Delezoide A-L, et al. Molecular characterization of early human T/NK and B-lymphoid progenitor cells in umbilical cord blood. Blood 2004;104:3918e26. 39. Gilliland D. Molecular genetics of human leukemias: new insights into therapy. Semin Hematol 2002;39:6e11.