Regulation of Development of CD56brightCD11c+ NK-like Cells with Helper Function by IL-18

Human γδ T cells augment host defense against tumors and infections, and might have a therapeutic potential in immunotherapy. However, mechanism of γδ T cell proliferation is unclear, and therefore it is difficult to prepare sufficient numbers of γδ T cells for clinical immunotherapy. Recently, natural killer (NK)-like CD56brightCD11c+ cells were shown to promote the proliferation of γδ T cells in an IL-18-dependent manner. In this study, we demonstrated that the NK-like CD56brightCD11c+ cells could directly interact with γδ T cells to promote their sustained expansion, while conventional dendritic cells (DCs), IFN-α-induced DCs, plasmacytoid DCs or monocytes did not. We also examined the cellular mechanism underlying the regulation of CD56brightCD11c+ cells. CD14+ monocytes pre-incubated with IL-2/IL-18 formed intensive interactions with CD56intCD11c+ cells to promote their differentiation to CD56brightCD11c+ cells with helper function. The development of CD56brightCD11c+ cells was suppressed in an IFN-α dependent manner. These results indicate that CD14+ monocytes pretreated with IL-2/IL-18, but neither DCs nor monocytes, play a determining role on the development and proliferation of CD56brightCD11c+ cells, which in turn modulate the expansion of γδ T cells. CD56brightCD11c+ NK-like cells may be a novel target for immunotherapy utilizing γδ T cells, by overcoming the limitation of γδ T cells proliferation.


Introduction
Human cd T cells recognize pathogens and autologous stress antigens and are involved in stress surveillance responses and maintenance of homeostasis in hosts [1,2]. They belong to the innate immune system and regulate acquired immunity through cytokine production and antigen presentation [3][4][5][6]. Because cd T cells distinguish infected cells and cancer cells from normal cells by detecting stress-induced molecules using cd T cell receptors (TCRs) and natural killer (NK) cell receptors, stimulation of cd T cells has gained attention as a potential therapeutic intervention for infections and malignancies [7][8][9][10][11][12]. However, cancer immunotherapy targeting cd T cells has met with limited success because of the difficulty of inducing the expansion of cd T cells in some cancer patients.
cd T cells are effectively activated by small foreign and self metabolites such as (E)-4-hydroxy-3-methylbut-2-enyl diphosphate and isopentenyl diphosphate in a classical MHC and MHCrelated molecule-independent manner [13,14]. It is important to note, however, that CD14 + antigen-presenting cells are required for the recognition of metabolite antigens by cd T cells, yet the precise mechanism for the recognition at the molecular level remains unclear [15]. In addition, the involvement of other immune cells such as NK cells and dendritic cells (DCs) in this recognition process has not been thoroughly explored [16][17][18][19][20][21].
It was previously demonstrated that human peripheral NK cells activated by Mycobacterium tuberculosis augmented the proliferation of cd T cells [22]. Peripheral blood DCs expressing CD56, an NK cell marker, promoted Th1-type responses of cd T cells stimulated by bisphosphonate and IL-2 [23]. We previously observed that CD56 bright CD11c + cells were involved in the IL-18-mediated expansion of cd T cells stimulated by IL-2 and zoledronic acid (ZOL) [24,25]. In addition, it was demonstrated that IL-18induced NK cells exhibited helper functions in the development of cytotoxic T lymphocytes (CTLs), although whether these NK cells also acted on cd T cells is yet to be determined [26,27].
IL-18 was originally identified as an IFN-c-inducing factor that activates NK cells [28]. Recent studies showed that IL-18 is produced by a wide variety of cells including non-immune as well as immune cells and the physiological roles of IL-18 extend far beyond serving merely as a cytokine inducer. For example, IL-18 is involved in angiogenesis [29] and metabolic syndromes [30,31]. Therefore, it is necessary to determine the various functions of IL-18 to clarify its central, biological and pathophysiological roles. IL-18 is produced as an inactive precursor and converted to an active form by the catalytic action of the inflammasome, which is composed of NLRP3, ASC, and caspase-1. Because it is activated by various stresses such as oxidation [32], IL-18 is considered to be one of the stress-sensing molecules. As IL-18 activates intracellular signals related to cell viability in NK cells [33] and memory-type CD8 + T cells [34] it is likely that IL-18 promotes proliferation and differentiation of certain cells expressing IL-18 receptors through activation of survival signals.
It was previously reported that IFN-a promoted the differentiation of monocytes to IFN-a-DCs that promote the generation of CD8 + CTLs, in addition to its anti-viral properties [35][36][37]. Several studies also indicated that IFN-a might activate cd T cells during infection [38][39][40]. In the present study, we examined how the development and proliferation of novel NK-like CD56 bright CD11c + cells were differentially regulated by CD14 + monocytes under the influence of IL-2/IL-18 or other cytokines including IFN-a, which will hopefully contribute to our understanding of the mechanisms behind the efficient expansion of human cd T cells.

Cell separation and culture
Peripheral blood mononuclear cells (PBMCs) were purified from heparinized blood of healthy human donors (ranging from 25 to 40 years of age) by Ficoll-Hypaque gradient centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) after receiving institutional review board approval (The Ethics Review Board of Hyogo College of Medicine, No.1033-2011) and written informed consent. PBMCs were cultured in AlyS505N-O medium (Iscove's MEM-based serum-free medium, Cell Science & Technology Institute, Inc., Sendai, Miyagi, Japan) supplemented with streptomycin, penicillin, glutamate, and 5% AB serum at 37uC in a humidified atmosphere containing 5% CO 2 . Depletion of CD3 + , CD56 + , or CD14 + cells from PBMCs was conducted using LD columns and microbeads conjugated with their specific mAbs (Miltenyi Biotec Inc.). Purification of cd T cells, CD56 + , CD14 + , and plasmacytoid DC (pDC) cells was carried out by positive selection using MS columns (Miltenyi Biotec Inc.). Cell isolation was performed according to the manufacturer's instructions. Growth factors, stimulators, and inhibitors were added to cultures at the following concentrations: 1 mM ZOL, 100 mM for 2M3B1PP, 10 ng/ml for IL-2, 100 ng/ml for IL-18, 5 ng/ml for GM-CSF, 20 ng/ml for IL-4, 1,000 U/ml for IFN-a, and 2.5 mg/ml for anti-IL-18Ra mAb, unless otherwise noted. Cell viability was determined by trypan blue dye exclusion or propidium iodide staining, and the cell number was calculated based on flow cytometry and cell viability. Cellular surface markers were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). CD14 + , CD56 int CD11c + , CD56 int CD11c 2 , CD56 bright CD11c + , and CD56 bright CD11c 2 cells were purified using a FACSAria cell sorter (Becton Dickinson) according to the manufacturer's instructions.

Expansion of cd T cells and CD56 bright CD11c + cells
For the expansion of cd T cells, PBMCs were stimulated with ZOL/IL-2/IL-18 at 1-5610 5 cells/well in a 48-well plate. CD56 bright CD11c + cells were expanded in culture of PBMCs or CD3 + T cell-depleted PBMCs at 1-5610 5 cells/well in a 48-well plate in the presence of IL-2/IL-18.

Preparation of DCs
For preparation of DCs, purified monocytes were incubated with GM-CSF/IFN-a or GM-CSF/IL-4 for 3-4 days in a 24-well plate at a cell concentration of 5610 5 /well. The resulting DCs (IFN-DCs or IL-4-DCs) were examined for the expression of CD56, CD11c, CD14, and CD62L. pDCs were purified (.95%, T cells (black line), and ab T cells (gray line). Data show mean 6 SD (n = 10), **p,0.01. (C) Requirement of CD56 bright CD11c + cells for maximal sustained proliferation of cd T cells. PBMCs were pre-stimulated with ZOL/IL-2/IL-18, and harvested on day 7. The proliferating cells were divided into 2 groups: one group was incubated with anti-CD56 antibody-conjugated beads and CD56 + cells were selectively removed. The other group was incubated with mouse IgG1-conjugated beads and used as a control. Both groups were re-incubated with ZOL/IL-2/IL-18 for another 14 days. Data show mean 6 SD (n = 5), **p,0.01. (D) Development of CD56 bright CD11c + cells and gradual reduced expression of CD11c. The number and CD11c expression of proliferated cells were analyzed by flow cytometry during the culture of CD3 + T cell-depleted PBMCs. (Grey shadow: isotype control; blue: CD56 int CD11c + cells on day 0; red: CD56 bright CD11c + cells on day 7; thin black line: day 14; and bold black line: day 21). Data show mean 6 SD (n = 5). A histogram shown is a representative of five independent experiments. (E) Comparison among CD56 bright CD11c + cells, monocytes, and several subsets of DCs in helper activity for cd T cells proliferation. Freshly isolated cd T cells(56104/well) were labeled with CFSE and co-cultured for 7 days with fresh CD14 + monocytes, IFN-a-DCs, IL-4-DCs, CD56 bright CD11c + , or pDCs, at a ratio of 1:1 in the presence of ZOL/IL-2/IL-18. Then, proliferative responses of cd T cells were analyzed based on flow cytometry and trypan blue dye exclusion test. R1: cd T cells labeled with CFSE undergoing cell division; R2: Unlabeled CD56 bright CD11c + cells. Data show mean 6 SD (n = 5), **p,0.01. The result is representative of five independent experiments. (F) Differential cytotoxicity of CD56 bright CD11c + cells against normal osteoblast cells (NHOst) and tumor cells (MG-63). The cytotoxicity was assessed using standard propidium iodide staining. Dot plots shown are representative of three independent experiments. doi:10.1371/journal.pone.0082586.g001 defined by flow cytometry) via positive selection with a CD303 + pDC Isolation kit.

Cell morphology
Cells were cultured in 24-well plates (Corning Inc., Corning, NY) and observed under a Nikon Digital Sight-Mac6000

Confocal microscopic analysis
PBMCs derived from a donor whose initial Vd2 + T cell frequency was 12.9% were incubated in the presence of ZOL/IL-2 for 10 days. The resulting cells (Vd2 + T cells .99%) were stained with CellTracker TM Green CMFDA (Life Technologies Corp., Carlsbad, CA) according to the manufacturer's protocol. For preparation of CD56 bright CD11c + cells, PBMCs were incubated with IL-2/IL-18 for 4 days and CD3 + T cells were removed using a CD3 Isolation kit and an AutoMACS Pro cell separator (Miltenyi Biotec Inc.). The CD3 2 cells were incubated for an additional 5 days in the presence of IL-2 and IL-18 and CD3 + T cells were removed. After incubation for an additional day, the CD56 bright CD11c + cells were harvested and stained with Cell-Tracker TM Red CMTPX (Life Technologies Corp.) according to the manufacturer's protocol. The green-and red-stained cells were mixed and placed in 35-mm glass base dishes (Glass 27w) (Asahi Glass Co., Ltd., Tokyo, Japan). After incubation with or without ZOL for 4 h, the cells were observed under an LSM 710 Laser Scanning Microscope (Carl Zeiss AG, Oberkochen, Germany) and the images were analyzed using Zen software (Carl Zeiss AG).

Cell Division Assay
CD56 + cells were enriched from PBMCs by depleting CD3 + and CD14 + cells. Monocytes were purified using a human monocyte enrichment kit. The enriched CD56 + cells were gently mixed with 3 mM carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFSE; molecular probe C-1157), and allowed to stand at room temperature for 15 min. An equal volume of 100% fetal bovine serum was added and incubated for 15 min at 37uC to remove excess CFSE. Labeled CD56 + cells and purified CD14 + monocytes were mixed at a ratio of 1:1 at a cell density of 2610 5 / well, incubated for 4 days, and then analyzed for the expression of CFSE, CD56, and CD11c.

Transwell test
Transwell test was performed using Costar transwell chambers with a pore size of 5 mm for cell penetration tests, and a pore size of 0.4 mm for soluble factor permeability tests. Purified CD14 + monocytes (.98% purity) were placed in the lower chambers and CD56 + cells (.95% purity) in upper chambers. After incubation for 5 days, the number of cells in the lower chambers was counted and the surface expression of CD14, CD56, and CD11c was analyzed by flow cytometry.

Statistical analysis
Statistical analysis was performed using Student's t-test or Bonferroni multiple comparisons test and expressed as the means 6 SD. Values of * p,0.05, or **p,0.01 were considered statistically significant.

Comparison of IL-18-induced CD56 bright CD11c + cells and conventional DCs on the helper function on cd T cells
Although we previously demonstrated that IL-2/IL-18-induced CD56 bright CD11c + cells enhanced ZOL-mediated expansion of cd T cells, the mechanism underlying the regulation of these cells in the culture of PBMCs with ZOL/IL-2/IL-18 has not been fully clarified. Consistent with our previous report, 1,4% and approximately 2% of freshly isolated PBMCs exhibited CD56 int CD11c + and Vd2 + phenotypes, respectively ( [25], data not shown). When PBMCs were incubated in the presence of ZOL/IL-2/IL-18 for 10 days, the number of CD56 int CD11c + cells was decreased in the CD3 2 gated area. In contrast, CD56 bright CD11c + cells appeared and eventually represented 10,18% of the total cells in the culture [25]. Whereas CD56 bright CD11c + cells were CD80/86 high , NKG2D high , and HLA-DR high , CD56 int CD11c + cells were CD80/86 low , NKG2D low , and HLA-DR low , as previously reported [25]. On day 15, however, the intensity of CD11c on these cells was reduced and CD56 bright CD11c 2 cells formed the majority phenotype found among CD3 2 cells (data not shown).
Whereas both CD56 bright CD11c + cells and cd T cells markedly proliferated in response to IL-2/IL-18/ZOL, the expansion of cd T cells was preceded by the development and proliferation of CD56 bright CD11c + cells in terms of absolute number (Fig. 1A). Consistent with previous findings, the majority of freshly isolated PBMCs were ab T cells, while CD56 int CD11c + and cd T cells constituted a minor population ( Fig. 1A; left). In addition, a small number of CD14 + CD11c + monocytes and CD56 + CD11c 2 NK cells were also present (data not shown). Stimulation of PBMCs by IL-2/IL-18/ZOL resulted in the development and expansion of CD56 bright CD11c + cells by around day 3 and they gradually increased the number thereafter. In contrast, cd T cells began to proliferate at around day 4 and their absolute number and proportion surpassed those of CD56 bright CD11c + cells by day 7 (Fig. 1B). It is worthy of note that the frequency of CD56 bright CD11c + cells was almost negligible in freshly isolated PBMCs. CD56 bright CD11c + cells were, however, multiplied up to nearly 10-fold and 200-fold the initial number of CD56 int CD11c + cells, a putative precursor of CD56 bright CD11c + cells, at day 3 and day 7, respectively. On the other hand, cd T cells apparently did not increase in number until day 3 or 4 at which point they rapidly increased and multiplied about 500-fold by day 7 (Fig. 1B). Although freshly prepared PBMCs contained ab T cells as a major population, the number was declined gradually, with CD4 + ab T cells being barely detectable by day 7 in particular (data not shown). CD14 + CD11c + monocytes, which initially constituted 5-10% of PBMCs, completely disappeared by day 7 (data not shown).
As previously reported, when CD14 + , CD56 + , or CD11c + cells were removed at the beginning of cell culture, the expansion of cd T cells in PBMC cultures was significantly impaired even in the presence of IL-2/IL-18 [25]. Co-culture of IL-2/IL-18-induced CD56 bright CD11c + cells with freshly isolated cd T cells, on the contrary, resulted in efficient expansion of cd T cells in the presence of ZOL/IL-2/IL-18 [25]. Thus, CD56 bright CD11c + cells are a prerequisite for the efficient expansion of cd T cells by day 7 of culture. In the present study, CD56 bright CD11c + cells were further depleted from ZOL/IL-2/IL-18-treated PBMCs at day 7 of culture by CD56 negative selection. The culture process of the resulting cells was re-started in the presence of ZOL/IL-2/IL-18 for another 14 days, and compared with that of consecutively cultured cd T cells. As shown in Fig. 1C, proliferation of cd T cells was significantly suppressed by removal of CD56 + cells although sustained partly. Thus, it was suggested that CD56 bright CD11c + cells were not continually essential during or after the logarithmic phase of cell expansion. However, the proficient supporting function of CD56 bright CD11c + cells in the early stage of culture was important for the maximal proliferation of cd T cells (Fig. 1C).
The generation of CD56 bright CD11c + cells appeared to be independent of CD3 + T cells, as they developed in the culture of CD3 + T cell-depleted PBMCs supplemented with IL-2 and IL-18 (Fig. 1D). Disappearance of CD11c was confirmed by flow cytometry during the late stage of culture period (by day 21). The number of CD56 int CD11c + cells, a possible precursor of CD56 bright CD11c + cells, were present at the frequency of 4-5% in the initial culture, disappeared from the culture soon after the beginning of culture (data not shown). In the absence of T cells, CD56 bright CD11c + cells progressively increased in both frequency and absolute number up to day 14. Thereafter, the intensity of CD11c levels on CD56 bright CD11c + cells progressively decreased during the culture (Fig. 1D).
We next examined whether CD56 bright CD11c + cells generated in the culture of T cell-depleted PBMCs were able to help development and expansion of cd T cells, comparing with various subsets of DCs or CD14 + monocytes. Freshly isolated CD14 + monocytes (.98%, purified by positive selection using MS column) were stimulated with IL-4/GM-CSF, or IFN-a/GM-CSF to induce IL-4-DCs or IFN-a-DCs. CD56 bright CD11c + cells were generated from the mixed culture of CD14 + monocytes and CD56 int CD11c + cells (selectively collected by FACS Aria cell sorter) in the presence of IL-2/IL-18, and CD303 + pDCs were purified by positive selection using MS columns. As shown in Fig. 1E, CD56 bright CD11c + cells strongly promoted the expansion of freshly isolated cd T cells in the presence of ZOL/IL-2/IL-18. In contrast, monocytes, IFN-a/GM-CSF-induced DCs, IL-4/ GM-CSF-induced DCs, and CD303 + pDCs failed to support the proliferation of cd T cells (Fig. 1E).

Interaction between cd T cells and CD56 bright CD11c + cells
Purified cd T cells (more than 98% pure) were incubated with CD56 bright CD11c + cells ( Fig. 2A, upper panel, left), ZOL-pulsed CD56 bright CD11c + cells (center), or ZOL-pulsed CD56 bright CD11c + cells with ZOL (right), and the cell cultures were observed under an optical microscope. ZOL-pulsed CD56 bright CD11c + cells formed larger aggregates than those without pulsing. To determine whether cd T cells directly interacted with CD56 bright CD11c + cells in the cell aggregates, both cells were separately stained with either green or red fluorescent dye respectively and observed under a confocal microscopy after incubation in the presence of ZOL ( Fig. 2A,  lower panel). cd T cells (green) and CD56 bright CD11c + cells (red) formed aggregates together and appeared to interact with each other. Similar results were obtained in the absence of ZOL (data not shown). We next examined the effect of ZOL on the regulation of cd T cell proliferation by CD56 bright CD11c + cells. Enriched CD56 bright CD11c + cells (1610 5 /0.5 ml/well) prepared from CD3 + cells-depleted PBMCs were pulsed with or without ZOL for 2 hours before co-culture with purified cd T cells (1610 5 / 0.5 ml/well). As shown in Fig. 2B, cd T cells alone slightly reduced their number during culture (from 1610 5 to 0.760.42610 5 ), and CD56 bright CD11c + cells strongly promoted expansion of cd T cells (from 1610 5 to 3.660.44610 5 ). Pulsing of CD56 bright CD11c + cells with ZOL significantly enhanced their helper function in proliferation of cd T cells, both in percentage (without pulsing:46%; with pulsing:63%) and in absolute number (without pulsing: 3.660.44610 5 ; with pulsing: 5.4260.69610 5 ). It was also shown that antigenic stimulation by ZOL could rather augment cd T cell expansion (6.2561.33610 5 ). Because freshly isolated cd T cells failed to proliferate significantly in response to IL-2, IL-2/ ZOL, or IL-2/2M3B1PP in the absence of accessory cells, even when IL-18 was present in the culture (Fig. 2C), it was suggested that IL-18-induced CD56 bright CD11c + cells played essential roles in the efficient expansion of cd T cells.
Although stimulation of purified cd T cells with either ZOL or 2M3B1PP alone did not alter the expression of co-stimulatory molecules such as CD28 and CD40L, addition of IL-2 either induced or augmented the expression of co-stimulatory molecules, CD28 and CD40L, and chemokine receptors, CCR5 and CCR7 (Fig. 2D). CD56 bright CD11c + cells produced CCL21, a chemokine that binds to CCR7, in an IL-2/IL-18-dependent manner (Fig. 2E). These results strongly suggest that IL-2/IL-18 may facilitate the interaction between cd T cells and CD56 bright CD11c + cells through co-stimulatory ligands/receptors, chemokine molecules/receptors, and adhesion ligands/receptors, leading to large cell aggregates and enhanced proliferation of cd T cells.
Requirement of IL-18-induced CD14 + monocytes for the development of CD56 bright CD11c + cells Next, we determined which types of cells were responsible for the development and expansion of CD56 bright CD11c + cells. CD14 + , CD56 + , or CD11c + cells were further removed from CD3 + T cell-depleted PBMCs, and the resulting cells were stimulated with IL-2/IL-18 for 7 days. The development of CD56 bright CD11c + cells in the culture was significantly impaired by the removal of these cells (Fig. 3A). In addition, cell division analyses using CFSE-labeling further suggested that CD56 bright CD11c + cells were derived from CD56 + cells and vigorously proliferated during cell culture (Fig. 3B). In contrast, CFSE-labeled CD14 + cells failed to proliferate. In addition, IL-2/ IL-18 failed to develop CD56 bright CD11c + cells in the culture of freshly isolated CD14 + monocytes (.98%, purified by positive selection using MS column) or CD56 int CD11c + cells alone (selectively collected by FACs Aria cell sorter), whereas CD56 bright CD11c + cells were generated in the mixed culture of CD14 + monocytes and CD56 int CD11c + cells in the presence of IL-2/IL-18, resulting in extensive cell aggregation (Fig. 3C). Next, the effect of various cytokines on CD14 + monocytes in the generation of CD56 bright CD11c + cells was examined (Fig. 3D). Freshly isolated CD14 + cells were pretreated with GM-CSF/IL-4, IL-2/ IL-18, and GM-CSF/IFN-a for 3 days to induce IL-4-DCs, IL-18-primed CD14 + monocytes, or IFN-a-DCs, respectively. Then, freshly isolated CD56 int CD11c + cells were added to the cultures (1610 5 cells/well) at a ratio of 1:1. After 12 h incubation, cells were observed under a microscope. As shown in Fig. 3D, cell aggregates appeared in the co-culture of CD14 + monocytes pretreated with IL-2/IL-18 and CD56 int CD11c + cells, but GM-CSF/IL-4-induced conventional DCs or GM-CSF/IFN-a-induced DCs failed to form cell aggregates when cultured with CD56 int CD11c + cells. Flow cytometric analyses of the expanded cells were carried out after 3 days of co-culture. There was a marked difference in the expression of CD11c and CD56 among cells. This suggested that stimulation of CD14 + monocytes by IL-2/IL-18 was essential for the development of CD56 bright CD11c + cells from CD56 int CD11c + cells (Fig. 3C and D).
Effect of IL-18 on CD14 + monocytes involved in the development of CD56 bright CD11c + cells As described above, CD56 bright CD11c + cells were generated in cultures of purified CD56 int CD11c + cells and CD14 + monocytes in the presence of IL-2/IL-18. Stimulation of CD14 + monocytes by IL-2, IL-18, or IL-2/IL-18 did not induce the proliferation. Cell division analysis using CFSE-labeled CD14 + monocytes also revealed that cellular division was not induced even in the presence of IL-2/IL-18, and the cells appeared to undergo apoptosis in the absence of these cytokines (Fig. 4A).
In order to address the possibility that CD14 + monocytes play a critical role in the IL-2/IL-18-mediated development of CD56 bright CD11c + cells from CD56 int CD11c + cells, we determined the phenotype of CD14 + monocytes. Freshly isolated CD14 + monocytes expressed IL-18 receptor aand b-chains, LFA-1, HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD54 (ICAM-1) (Fig. 4B and 4C). They had enhanced expression of HLA-ABC, HLA-DR, CD80, CD40, CD54, and CD209 after incubation with IL-2/IL-18 for 24h (Fig. 4C). To examine how monocytes are involved in the development of CD56 bright CD11c + cells from CD56 int CD11c + cells, transwell tests were performed according to the method described in Material and Methods. To analyze cellular interactions between CD56 int CD11c + cells and CD14 + monocytes, transwell chambers with a pore size of 5 mm for cell penetration tests and those with a pore size of 0.4 mm for soluble factor permeability tests were employed (Fig. 4D). Purified CD14 + monocytes (.98% purity) were placed in the lower chambers and CD56 + cells (.95% purity) containing CD56 int CD11c + cells in the upper chambers. After incubation for 5 days in the presence of IL-2/IL-18, the number of cells in lower chambers was counted and the frequency of cells in the lower chambers was analyzed by flow cytometry about CD56 and CD11c expression. Experiments demonstrated that soluble factors alone failed to induce CD56 bright CD11c + cells from CD56 int CD11c + cells, and that cell-cell contact between CD14 + monocytes and CD56 int CD11c + cells was critical for the development of CD56 bright CD11c + cells (Fig. 4D). In accordance with this observation, cell aggregates appeared in co-cultures of CD14 + monocytes with CD56 int CD11c + cells (Fig. 3C, D). Taken together, these results suggest that stimulation of CD14 + monocytes by IL-2/IL-18 is essential for the development of CD56 bright CD11c + cells via intensive cell-cell interactions.
Inhibition of CD14 + monocytes-dependent development of CD56 bright CD11c + cells by IFN-a Because various cytokines affect differentiation and function of monocytes, we next examined the mechanism by which cytokines could modulate the functions of CD14 + monocytes in the development of CD56 bright CD11c + cells from CD56 int CD11c + cells. IFN-a is known to facilitate the differentiation of monocytes to IFN-a-DCs that activate CD8 + CTLs (35)(36)(37). When PBMCs were stimulated with ZOL/IL-2, the proliferation of cd T cells was abrogated by IFN-a in a dose-dependent manner (Fig. 5A). Whereas IL-18 significantly promoted the expansion of cd T cells in the culture of PBMCs stimulated by ZOL/IL-2, the addition of IFN-a reduced such expansion (Fig. 5B).
In addition, IFN-a also inhibited the development of CD56 bright CD11c + cells in cultures of CD3 + T cell-depleted PBMCs stimulated with IL-2/IL-18 (Fig. 5C), Furthermore monocytes primed with IL-2/IL-18 in the presence of IFN-a failed to induce generation of CD56 bright CD11c + cells leading to inhibition of expansion of cd T cells (Fig. 5D). CD14 + monocytes (2610 5 cells/well) were incubated with or without IFN-a for 3 days in the presence of IL-2/IL-18, then CD56 int CD11c + cells (2610 5 cells/well) were added to the culture. The morphological difference of cells in co-culture was observed by microscopy after additional 3 days (upper panels). Further, freshly isolated cd T cells (2610 5 cells/well) were added to the culture and incubated of CD56 int CD11c + cells and CD14 + monocytes, in the absence and presence of freshly isolated cd T cells. CD14 + monocytes were pretreated with IL-2/IL-18 for 3 days, with or without IFN-a then CD56 int CD11c + cells were added into the culture (upper panels). Next, freshly isolated cd T cells were added and cellular clusters were observed by microscope (lower panels). The cell aggregates image is representative of three independent experiments. (E) Proliferation of cd T cells in cultures containing mature CD56 bright CD11c + cells even in the presence of IFN-a. Freshly isolated cd T cells and mature CD56 bright CD11c + cells were stimulated with ZOL/IL-2, with or without further addition of IFN-a since day 7 onwards and were continuously incubated. The number of proliferating cells was assayed after another 7 days' culture. Data show mean 6 SD (n = 5). doi:10.1371/journal.pone.0082586.g005 for another 3 days. Large clusters were observed in the culture incubated with IL-2/IL-18 alone, but not in the culture added with IFN-a (lower panels). Thus IFN-a was suggested to alter the effect of IL-2/IL-18 on CD14 + monocytes resulting in inhibition of development of CD56 int CD11c + cells to CD56 bright CD11c + cells.
Furthermore, it is of note that the expansion of cd T cells was not diminished by the addition of IFN-a when freshly isolated cd T cells were co-incubated with mature CD56 bright CD11c + cells (Fig. 5E). Freshly isolated cd T cells and mature CD56 bright CD11c + cells (purified from IL-2/IL-18-pretreated CD3 + depleted PBMCs for 7 days) were co-cultured with ZOL/ IL-2, with or without further addition of IFN-a since day 7 onwards and were continuously incubated. The number of proliferating cells was assayed after another 7 days' culture. Proliferation of cd T cells appeared not to be influenced by IFN-a. These results suggest that IFN-a abrogated the expansion of cd T cells by inhibiting the IL-2/IL-18-mediated development of mature CD56 bright CD11c + cells from CD56 int CD11c + cells, rather than directly inhibiting the growth of cd T cells.

Discussion
Evidence is accumulating that human cd T cells are involved in the first line of defense against infections and malignancies. Although much attention has been paid to cancer immunotherapy using cd T cells, hyporesponsiveness of cd T cells to phosphoantigens has hampered the development of novel cd T cell therapy for cancer patients. Thus, it is important to clarify the cellular mechanisms underlying the expansion of cd T cells.
CD14 + monocytes can internalize nitrogen-containing bisphosphonates (N-BPs) such as ZOL and present IPP or IPP-related antigens to cd T cells. This fluid-phase endocytosis occurs predominantly in CD14 + monocytes at relatively low concentrations of N-BPs. Thus, CD14 + monocytes play an essential role in antigen presentation to cd T cells. Whereas CD14 + monocytes are pivotal in the initial activation of cd T cells, they fail to directly support the subsequent proliferation of cd T cells. The present study revealed that CD14 + monocytes not only functioned as APCs, but also were important for the development and proliferation of CD56 bright CD11c + cells that could directly act on cd T cells to positively regulate their expansion (Fig. 6).
Because CD56 bright CD11c + cells were generated in cultures of CD56 int CD11c + cells and CD14 + monocytes in the presence of IL-2/IL-18, it was likely that CD14 + monocytes induced the generation of CD56 bright CD11c + cells from CD56 int CD11c + cells. Treatment of CD14 + monocytes with ZOL inhibits, farnesyl diphosphate synthase, an enzyme in the isoprenoid pathway involved in the biosynthesis of isoprenoid metabolites, causing a low intracellular level of geranylgeraniol, which may be responsible for the activation of caspase 1 and the maturation of IL-18 [13][14][15][16][17][18]. Endogenous IL-18 is thus essential for the development of CD56 bright CD11c + cells, while other soluble factors or signaling pathways required for interactions between CD14 + monocytes and CD56 int CD11c + cells remains to be identified. Nascent CD56 bright CD11c + cells developed under the influence of IL-2/IL-18 expressed an intermediate level of CD11c, a high level of APC-related molecules such as CD80, CD86, HLA-DQ, and HLA-DR, and a high level of ICOS and CD25 [24,25]. These cells tended to aggregate among themselves without exogenous cytokines or chemokines. When they were mixed with cd T cells, they quickly formed large cell aggregates and an efficient proliferation of cd T cells was observed even in the absence of antigens. None of the other cell types, including monocytes, conventional DCs, IFN-a-induced DCs, and pDCs formed cell aggregates with cd T cells. This indicates that nascent CD56 bright CD11c + cells have a natural affinity to cd T cells and promote their proliferation in an antigen-independent manner. However, proliferation may be adhesion molecule-dependent because both cell types express adhesion molecules such as LFA-1 and ICAM-1. The addition of ZOL further enhanced the proliferation of cd T cells demonstrating that CD56 bright CD11c + cells can also serve as APCs, while the efficiency for fluid-phase endocytosis of ZOL might be lower than that of CD14 + monocytes. During the course of cell culture, CD56 bright CD11c + cells tended to concomitantly lose CD11c expression and the ability to support cd T cell expansion. The supporting function of CD56 bright CD11c + cells reached a peak between 3 and 7 days after the start of cell culture. When CD11c expression was completely lost between day 15 and day 21, they also lost their supporting function. CD11c by itself, however, might not be involved in the helper function, because other CD11c-highlyexpressing cells failed to both form cell aggregates and enhance cd T cell proliferation.
Consistent with our previous reports, CD56 bright CD11c + cells showed a high level of tumoricidal activity in an effector-to-target ratio-dependent manner. In this study, we further examined the specificity of the cells. It is of note that CD56 bright CD11c + cells could recognize and lyse malignant tumor cells, distinguishing them from normal cells. This finding provides compelling evidence to support the classification of CD56 bright CD11c + cells as NKlineage cells. Because both NK-lineage cells and cd T cells belong to innate immune cells, they may interact and serve as a bridge between the innate and adaptive immune responses. Indeed, both CD56 bright CD11c + cells and cd T cells express high levels of APCrelated molecules and may present peptide antigens derived from lysed tumors and infected cells to conventional ab T cells.
This study demonstrated that CD14 + monocytes could induce the development of NK-like CD56 bright CD11c + cells under the influence of IL-2/IL-18 and positively regulate the proliferation of cd T cells by an indirect mechanism. Because many reports have suggested interactions between NK cells and monocytes/DCs, which may determine the differentiation and proliferation of distinct subsets of NK cells, we next attempted to determine the molecular mechanism that negatively modulated the development of CD56 bright CD11c + cells and subsequently impaired cd T cell expansion.
We demonstrated that cell-cell contact was important for the development of CD56 bright CD11c + cells. Neither purified CD14 + cells nor CD56 int CD11c + cells alone expanded to CD56 bright CD11c + cells. Therefore, not only soluble factors such as cytokine, and cellular interaction between CD14 + cells and CD56 int CD11c + cells were necessary for CD56 bright CD11c + cells generation, as confirmed by transwell and co-culture tests. IL-18 prolonged the viability of CD14 + monocytes, and up-regulated the expression of co-stimulatory molecules such as HLA-ABC, HLA-DR, CD80, CD40, and ICAM. Because freshly isolated CD14 + monocytes expressed both Ra and Rb chains of IL-18R, IL-18 may directly transduce signals to monocytes. Recently, M-CSFstimulated monocytes were shown to express membrane-bound IL-18 [41,42], although the physiological roles and functions of these monocytes in inflammation remain to be explored. It may be of interest to examine the relationship between IL-18-stimulated monocytes and IL-18-expressing monocytes. Further study is required to clarify the development, phenotypes, and roles of these monocytes.
It is generally difficult to expand cd T cells from PBMCs that contain a low frequency of cd T cells, compared with PBMCs that contain a high frequency of cd T cells. When PBMCs with a low cd T cell frequency are stimulated with ZOL/IL-2, many adherent cells can be seen by microscope. In contrast, only large cell aggregates are present when PBMCs with a high cd T cell frequency are used. This suggests that to expand subsets of effector cells the corresponding APCs are necessary as supporting cell types. For example, conventional DCs and IFN-a-DCs support the development of CD8 + CTL cells, and PGE-2-DCs plays role in the augmentation of regulatory T cells. In the case of PBMCs with a low cd T cell frequency, pDCs survived and inhibited the generation of CD56 bright CD11c + cells, leading to the impaired proliferation of cd T cells (data not shown). Taken together, this suggests that IL-18 may activate CD14 + monocytes to facilitate the expansion of both CD56 bright CD11c + cells and cd T cells. Furthermore, it is worth noting that IFN-a failed to interfere with the functions of mature CD56 bright CD11c + cells, because the addition of mature CD56 bright CD11c + cells to the in vitro culture system overcame the inhibitory effect of IFN-a.
Although IL-18 was originally discovered as an IFN-c-inducing factor, its physiological roles have not been fully clarified. The present study demonstrated that IL-2/IL-18 facilitated the development of CD56 bright CD11c + cells by CD14 + monocytes. Because activated cd T cells also express IL-18 receptor a and b chains, IL-18 may directly act on cd T cells. In addition, IL-18 has been shown to maximize innate immune responses of ITAMbearing lymphocytes and up-regulate Bcl-2 and Bcl-X L , which protect mitochondria and augment survival signaling. IL-18 was recently shown to prime NK cells to have unique helper activity, and the resulting ''helper'' cells promote activation of DC and DC-mediated recruitment of effector CD8 + T cells to the tumor microenvironment [43]. It is thus intriguing to compare the physiological function and roles of ''helper'' NK cells with the present CD56 bright CD11c + cells as well as murine IKDCs or NKDCs.
Based on the present results, signals transduced from TCR, costimulatory receptors, adhesion molecules, and IL-18 receptors are required for the full activation and sustained proliferation of cd T cells. In conclusion, CD14 + monocytes play a critical role in the generation of novel CD56 bright CD11c + cells that directly interact with activated cd T cells and sustain their robust expansion. This function of monocytes can be altered by IFN-a, which induces the differentiation of CD14 + monocytes to IFN-DCs (Fig. 6). The precise, physiological role of IL-18, CD14 + monocytes, and CD56 bright CD11c + cells in innate immune responses remains to be established.