Synergistic induction of interferon-γ by interleukin-2, interleukin-12 and poly(I:C) in a human natural killer cell line

Abstract Natural killer (NK) cells express various Toll-like receptors (TLRs). Little is known about the role of TLRs in direct NK cell activation. To clarify possible synergistic roles of cytokine and TLR signaling, human NK cell line KHYG-1 was stimulated with agonists for TLR1-9. IFN-γ production was not significantly induced following stimulation with single TLR agonists. Of the nine TLR agonists tested, only poly(I:C) strongly upregulated IFN-γ production by synergistic interleukin-2 (IL-2) and IL-12 stimulation. The role of TLR3 signaling was also examined. An inhibitor of Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) blocked this synergistic action. However, TLR3 expression was unchanged in the presence of IL-2 and IL-12. Our findings suggest a possible role for type 1 cytokines in NK cell IFN-γ production against viral infections.


Introduction
Natural killer (NK) cells are primitive immune cells, which trigger first-line defense against viral infection and neoplasms. In mucosal tissues, such as the intestinal and genital surfaces, localized NK cells often encounter pathogenic microorganisms including viruses, bacteriae and fungi. Generally accepted idea is these pathogens activate antigen-presenting cells (APCs) during innate immune responses. Following pathogen exposure, APCs recognize pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs), enabling them to secrete cytokines, such as Interleukin (IL)-12 [1].
Recently, NK cells have been demonstrated to express TLRs and to directly recognize PAMPs [2][3][4][5]. Although both human and murine NK cells have been reported to express the majority of TLRs, whether direct, ligand-mediated TLR activation occurs in NK cells remains controversial [6][7][8]. Of interest, the requirement of IL-12 for IFN-c production by TLR stimulation has been reported [5,9]. We and others have reported indispensable roles for IL-2 and IL-12 in the stimulation of lymphokineactivated killer (LAK) cells against choriocarcinoma and autologous tumor cells [10][11][12]. Therefore, we asked whether the presence of IL-2 and IL-12 would affect TLR3 expression and subsequent TLR3-mediated NK cell activation. In addition, the intracellular signaling of direct NK cell activation via TLR3 has not been revealed yet. Toll-IL-1 receptor domaincontaining adaptor inducing IFN-b (TRIF) is an adaptor mediating TLR3 signaling in innate immune cells. Thus, in order to investigate the involvement of TRIF, we used a TRIF inhibitor to block the TRIF pathway.
In the present study, we demonstrated that IL-2 and IL-12 enhanced poly(I:C)-induced IFN-c production in the human mucosal NK cell line KHYG-1 without enhanced TLR3 expression.

Flow cytometry
Fluorochrome-conjugated mouse monoclonal antibody (mAbs) against human CD69 was purchased from BD Biosciences (Franklin Lakes, NJ). The mouse mAb against human TLR3 was purchased from IMGENEX (San Diego, CA). Direct staining was performed by adding 20 mL of the mAbs to each sample and incubating the cells at 4 C for 30 min. KHYG-1 cells were fixed and permeabilized using BD Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer's instructions. Expression of TLR3 in KHHG-1 was determined by adding 5 mL of the anti-TLR3 antibody conjugated with PE at 4 C for 30 min. Flow cytometry was performed using CellQuest software on a BD FACSCalibur (BD Biosciences) instrument.

TLR agonists treatment
The KHYG-1 cells were incubated with or without TLR1-9 agonists (Human TLR1-9 agonist Kit, InvivoGen, San Diego, CA) in the presence or absence of IL-2 (10 5 U/mL) and IL-12 (Recombinant Human IL-12 (p70), BD Biosciences) (10 4 U/mL) for 24 h. The optimal concentrations suggested by the manufacturer were utilized throughout the study. Before these experiments, we examined the baseline mRNA expression of TLR1-9 in unstimulated KHYG-1 cells by RT-PCR as described previously (data not shown).

ELISPOT IFN-c detection
The number of IFN-c producing cells was examined using an ELISpot for Human Interferon-c Kit (MABTECH AB, Nacka Strand, Sweden); all protocols were performed according to the manufacturer's instructions. Briefly, the KHYG-1 cells that had been stimulated with the TLR1-9 agonists for 24 h with or without IL-2 and IL-12 were seeded at 1 Â 10 5 cells/well and incubated for 5 h on membranes coated with mouse anti-human IFN-c capture mAb. After washing, the plates were incubated with biotinylated anti-human IFN-c detection mAb and treated with streptavidin-ALP and BCIP/NBT substrate (Bio-Rad, Richmond, CA). The plates were then washed to stop the color development. The colored spots were counted using the ImmunoScan system (Cellular Technology Ltd., Shaker Heights, OH).

TRIF inhibitor administration
To examine the indispensable role of TLR3 on IL-2 þ IL-12-induced IFN-c production, KHYG-1 cells were treated with a TRIF inhibitor. KHYG-1 cells were seeded into 24-well plates (5 Â 10 5 cells/well) and incubated with 12.5 mg/mL poly(I:C) and 50 mM of the TRIF inhibitor peptide (Pepinh-TRIF, InvivoGen, San Diego, CA) for 24 h under presence or absence of IL-2 and IL-12. The optimal concentration of Pepinh-TRIF was suggested by the manufacturer and we confirmed Pepinh-TRIF had no cytotoxic effect on KHYG-1 cells by preliminary experiment (data not shown). The culture supernatants were collected and centrifuged at 3000g at 4 C for 5 min to remove cellular debris; samples were stored at À80 C until use.

IFN-c ELISA
The IFN-c levels in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) using the Human IFN-c ELISA Kit (Thermo, Rockford, IL), which was utilized according to the manufacturer's instructions. Briefly, 50 mL of the biotinylated antibody reagent and 50 mL of the IFN-c standards or culture supernatant samples were added to 96-well plates coated with the anti-IFN-c antibody and incubated for 2 h at room temperature. After the incubation, the plates were washed and incubated with 100 mL of streptavidin-HRP for 30 min at room temperature. After washing, the plates were incubated with the TMB substrate for 30 min. Reactions were stopped through the addition of 100 mL of stop solution. Sample absorbance was read at 450 nm using a microplate reader.

Statistical analysis
The data were analyzed using the Tukey-Kramer test and Welch's t-test using Statcel 2 software (OMS Publishing Inc, Tokorozawa, Saitama, Japan). A probability level of 5% (p < .05) was considered to be statistically significant.

TLR agonist stimulation is insufficient to induce KHYG-1 cell IFN-c production
We treated KHYG-1 cells with TLR1-9 agonists for 24 h under presence or absence of IL-2 and IL-12 and examined IFN-c production using a highly sensitive ELISPOT assay. IFN-c production was not significantly induced following TLR agonist stimulation without IL-2 or IL-12 for 24 h ( Figure  1). Under the combination of IL-2 and IL-12, of the nine TLR agonists, only poly(I:C) increased IFN-c production ( Figure 1). This finding suggests that IL-2 and IL-12 are essential for TLR3-mediated IFN-c production in KHYG-1 cells.

IL-2 and IL-12 do not affect KHYG-1 cell TLR3 expression
Recently, cytokine-induced upregulation of TLRs has been reported in various inflammatory cell types, including human neutrophils [14] and monocytes [15], murine monocytes [16] and mast cells [17]. However, whether cytokine-mediated induction of TLRs occurs in human mucosal NK cells remains unknown.

Synergistic upregulation of IFN-c production by IL-2/IL-12 and poly(I:C) is mediated through the TLR3/TRIF pathway
On the basis of the above results, we hypothesized that the synergistic upregulation of IFN-c by IL-2/ IL-12 and poly(I:C) in KHYG-1 cells is mediated by enhanced signaling through the TLR3/TRIF cascade. To test this hypothesis, we treated KHYG-1 cells with TRIF inhibitory peptides. As shown in Figure  3, the TRIF inhibitory peptide Pepinh-TRIF significantly suppressed the effects of IL-2 and IL-12 on poly(I:C)-induced IFN-c production.

Discussion
Natural killer cells are an important part of the innate immune system to kill tumors and virally infected cells without MHC restriction or previous antigen exposure [18]. NK cells also produce cytokines that contribute to early host defense responses against pathogens [19]. Mucosal NK cells, including uterine NK (uNK) cells, frequently contact pathogens and virus-infected cells. We have recently reported that IL-2 is important for lipopolysaccharide-induced IFN-c production by human decidual mononuclear cells [20]. However, because of the difficulty of maintaining freshly prepared decidual NK cells, a detailed analysis has not been possible. Thus, we employed the large granular human NK cell line KHYG-1 in this study as a model for mucosal NK cells. The KHYG-1 cell line was established from a patient with NK cell leukemia [13], and it has been employed as a human NK cell model in multiple studies [21][22][23][24][25].
Toll-like receptors are an evolutionarily conserved family of transmembrane proteins that play a primary role in innate immunity. In mammals, TLRs are expressed on mucosal innate immune cells that recognize PAMPs. TLR binding of PAMPs triggers the innate immune response through nuclear factor-jB (NF-jB)-dependent and interferon regulatory factor (IRF)-dependent signaling pathways [1]. TLRs play physiological roles in dermal and mucosal immune cells, which cope with multiple types of pathogens, including viruses.
In the present study, we asked whether NK cells use TLR3 to recognize PAMPs and to regulate cell function. We confirmed TLR3 expression in the NK cell line KHYG-1, implying that they are poised to rapid response to local viral exposure.
Of interest, though the TLR3 agonist poly(I:C) alone resulted in weak IFN-c production, simultaneous stimulation with IL-2 and IL-12 strongly induced IFN-c production. Importantly IL-2 and IL-12 treatment induced slightly upregulation of CD69 expression, TLR3 expression was unchanged. These findings indicate enhanced IFN-c production does not simply reflect NK cell activation. Our results suggest that the cytokine signals IL-2 and IL-12 may be essential for the regulation of direct activation of NK cells by TLR3, which increases the initial potency of these cells at the site of infection and promotes the rapid development of subsequent  adaptive immune responses. Our hypothesis was confirmed through experiments with TRIF inhibitory peptides. These results are partially consistent with our previous study employing primary human decidual mononuclear cell cultures that failed to detect IL-12-induced TLR4 upregulation [20]. However, decidual mononuclear cells contain various cell types including dendritic cells, macrophages and neutrophils. This fact possibly explains the discrepancy between the results obtained in this study and our previous work.
In conclusion, our results demonstrate that a NK cell line KHYG-1 constitutively express intracellular TLR3. IL-2 and IL-12 enhance TLR3 signaling in KHYG-1 cells to produce IFN-c. These findings suggest possible roles of type 1 cytokines for NK cells during mucosal inflammatory response against viral infections.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was supported by 'Strategic Research Base Development' Program for Private Universities subsidized by MEXT.