s2fieldsofstudy: Biology Medicine

text:
The Plasticity of Th17 Cells in the Pathogenesis of Rheumatoid Arthritis

Helper T (Th) cells play an important role in the pathogenesis of autoimmune diseases, including rheumatoid arthritis (RA). It has been revealed that Th17 cells can shift to Th1 cells (i.e., “nonclassic Th1 cells”), which are reported to be more pathogenic than Th17 cells per se. Thus, the association of Th cells in the pathogenesis of autoimmune disease has become more complicated. We recently reported using peripheral blood from untreated and early-onset RA patients that the ratio of CD161+Th1 cells (i.e., Th17-derived Th1 cells to CD161+Th17 cells) is elevated and that levels of interferon-γ (IFNγ)+Th17 cells are inversely correlated with levels of anti-CCP antibodies. Here, we review the plasticity of Th17 cells in the pathogenesis of RA, suggesting possible implications for novel therapies.

Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease with chronic joint inflammation and destruction, and is characterized by activated T cells [1]. In 1999, we reported that IL-17 from activated human T cells in the synovial tissues of RA patients is a potent stimulator of osteoclastogenesis [2]. Th17 cells have been reported to play important roles in the pathogenesis of RA [3,4] since their identification in 2005 [5] (Figure 1). In addition, it has been revealed that Th17 produce IL-21, IL-22, TNFα, except IL-17.

Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease with chronic joint inflammation and destruction, and is characterized by activated T cells [1]. In 1999, we reported that IL-17 from activated human T cells in the synovial tissues of RA patients is a potent stimulator of osteoclastogenesis [2]. Th17 cells have been reported to play important roles in the pathogenesis of RA [3,4] since their identification in 2005 [5] (Figure 1). In addition, it has been revealed that Th17 produce IL-21, IL-22, TNFα, except IL-17.  [5] and (b) macrophages to produce TNFα [6]; (c) IL-17 also stimulates osteoblasts to produce RANKL, potently Figure 1. The role of Th17 cells in the pathogenesis of rheumatoid arthritis (RA). Th17 cells play a central role in the pathogenesis: (a) IL-17 stimulates synovial fibroblasts to produce IL-6 [5] and (b) macrophages to produce TNFα [6]; (c) IL-17 also stimulates osteoblasts to produce RANKL, potently inducing osteoclastogenesis [2,3]; (d) In addition, IL-17 induces osteoclastogenesis from monocytes alone in the absence of osteoblasts or RANKL [7]; (e) RANKL and TNFα synergistically induce osteoclastogenesis [7]; (f) IL-6 induces differentiation of Th17 cells [8]; (g) TNFα is produced by Th17 cells per se; thus, Th17 cells differentiate to nonclassic Th1 in an autocrine or paracrine manner [9]; (h) RANKL expressed on the surface of Th17 cells converts nonresorptive osteoclasts to resorptive osteoclasts via cell-cell contact [10]. RANKL, receptor activator of nuclear factor kB.
Several groups have reported IL-17 as an important cytokine in the early phase or the disease-onset phase of RA. The peripheral level of IL-17 is significantly high in RA patients whose disease durations are less than 9 weeks [11]. In addition, the levels of IL-17 in individuals before RA onset is significantly higher than that in patients after RA onset [12]. In 2013, Chalan et al. reported that the number of circulating CD4+CD161+T lymphocytes are elevated in seropositive arthralgia before the onset of RA but decreased in patients with newly diagnosed RA [13]. In contrast, a regulatory variant in CC chemokine receptor 6 (CCR6, a specific marker for Th17 cells [14,15]) is related to RA susceptibility [16]. Thus, many findings support that IL-17 plays a crucial role in the disease onset or the early phase of RA.
Helper T (Th) cells play an important role in the pathogenesis of autoimmune diseases, including RA [17]. In 2005, Th17 cells were reported as a novel Th cell [5]. In addition, it was revealed that Th17 cells can shift to Th1 cells (i.e., "nonclassic Th1 cells"), which are reported to be more pathogenic than Th17 cells per se [18][19][20][21]. Thus, a novel dichotomy-classic Th1 cells and nonclassic Th1 cells-appeared [19,20]. The association of Th cells in the pathogenesis of autoimmune disease has become more complicated. In addition, numerous "Th1 cells" studies from before nonclassic Th1 cells were discovered may need to be evaluated again, because "Th1 cells" in the previous studies included both classic and nonclassic Th1 cells. We review the plasticity of Th17 cells in the pathogenesis of RA.

Plasticity of Th17
In 1986, Mosmann et al. reported the dichotomy of mouse helper T cells Th1 and Th2. Th1 cells produce interferon-γ (IFNγ), stimulating cellular immunity and protecting intracellular pathogens [22]. In contrast, Th2 cells produce IL-4, inducing humoral immunity. Based on this Th dichotomy, the "Th1/Th2 paradigm", autoimmune diseases were divided into two categories-"Th1 diseases" and "Th2 diseases". Rheumatoid arthritis (RA) was supposed to be a Th1 disease; however, both sensitive and specific radioimmunoassays and a standard cytopathic inhibition assay showed little or no IFNγ in synovial fluids [23][24][25]. Thus, in the 1990s, both clinical and basic researchers began to think that the "Th1/Th2 paradigm" was too simplistic.
In 1996, human IL-17 was cloned as a novel cytokine [5]. We demonstrated that IL-17 plays a crucial role in the pathogenesis of RA and osteoclastogenesis in 1999 [2]. (We call "IL-17A" "IL-17" in the current review.) In 2005, the 3rd Th cell type, Th17, was reported, which was firstly called "Th IL-17 " [26,27]. Th17 cells play a role in protective immunity against extracellular bacterial infection [28]. We also demonstrated that IL-17 induces human osteoclastogenesis even in the absence of osteoblasts [7] and that the IL-23-IL-17 axis plays an important role in mouse arthritis and human osteoclastogenesis [29]. It has been revealed that Th17 plays an important role in the pathogenesis of many autoimmune diseases including RA [5].
Th17 cells are highly unstable and easily shift to Th1 cells [33,34]. In addition, it has recently been reported that TGFβ and IL-6 induce Th1-to-Th17-cell transdifferentiation in the mouse gut, as a "reverse plasticity" [35]. The discovery of this alternative pathway of Th17-and Th17/Th1-cell generation is likely to have important implications for both immune-mediated diseases and protective immune responses, in particular in the gut [36].

Th17-Derived Th1 Cells (CD161+ Th1 Cells, Nonclassic Th1 Cells)
In 2008, Cosmi et al. reported that CD161 (or NKR-P1A) gene is up-regulated in human Th17 clones and that all IL-17-producing cells are contained in the CD161+ fraction of CD4+ T cells present in the circulation or in inflamed tissues [31]. More importantly, they showed that all IL-17-producing cells originate from CD161+ naïve CD4+ T cells of umbilical cord blood in response to the combined activity of IL-1β and IL-23 [31]. In 2009, Kleinschek et al. identified human CD161+ CD4 T cells as a resting Th17 pool that can be activated by IL-23 and mediate destructive tissue inflammation using blood and fresh colon specimens from patients with Crohn's disease [37]. Thus, these findings indicated that CD161 is a novel surface marker for human Th17 cells.
Two ligands have been identified for human CD161. One ligand for CD 161 was identified as the lectin-like transcript-1 (LLT-1) in 2005 [38]. It is suggested that CD161 plays an important role in favoring trans-endothelial migration of Th17 cells into tissues [31]. Another ligand of CD161 was identified as the proliferation-induced lymphocyte-associated receptor (PILAR) in 2008 [39]. In the absence of CD28 costimulation, PILAR signaling through CD161 supports T-cell proliferation by increasing the expression of antiapoptotic Bcl-xL and induces secretion of Th 1 cytokines [39]. In addition, PILAR was expressed by 10% of CD4+ T cells in synovial fluid from one patient with RA [39]. Thus, CD161 with these ligands plays important roles in both human immunity and the pathogenesis of RA.
In 2010, Nistala et al. showed that Th17/Th1 cells from the joints of children with inflammatory arthritis highly express both Th17 and Th1 lineage-specific transcription factors, receptor-related orphan receptor C 2 (ROR C 2) and T-bet [40]. They also showed that Th17 cells shift to Th17/Th1 with low TGFβ and high IL-12 levels, which mimics the condition of the disease site. In addition, Th17/Th1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin. They showed that synovial Th17 and Th17/Th1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. Thus, these findings provided evidence that the Th17 phenotype is unstable and that Th17 cells can shift to Th17/Th1 and Th1 cells in human arthritis.
In 2011, Cosmi et al. reported that CD4+CD161+T cells can shift from Th17 cells to the Th17/Th1 or Th1 phenotype in the SF of children with oligoarticular-onset JIA [18]. In addition, they also showed that the accumulation of these cells is correlated with parameters of inflammation. Thus, these findings support the hypothesis that these cells play a pathogenic role in JIA disease activity.
A pathogenic role of the nonclassic Th1 subset has been reported in human inflammation. Nonclassic Th1 is detected in tissue from JIA patients [18,40]. In 2014, Maggi et al. demonstrated that Th17 cells, which express TNFα, can shift to nonclassic Th1 cells in an autocrine or a paracrine manner [9]. In 2013, Maggi et al. reported that CD4+CD161+T cells infiltrate into Crohn's disease-associated perianal fistulas and that the number of CD4+CD161+ Th cells decreases with anti-TNF therapy, adalimumab [41]. In 2016, they also reported that nonclassic Th1 cells, but not Th17 cells, induce vascular cell adhesion molecule-1 (VCAM-1, CD106) in fibroblast-like synoviocytes from JIA [42]. Ramesh et al. demonstrated that Th17 cells expressing P-glycoprotein (P-gp) produce both Th17 cytokines and Th1 cytokine and that P-gp+ Th17 cells are refractory to the glucocorticoid used to treat clinical autoimmune diseases [43]. Martin-Orozco et al. reported that islet-reactive Th17 cells promote pancreatic inflammation, but only induce diabetes upon conversion into IFNγ producers using non-obese diabetic (NOD) scid mice and neonate NOD mice [44]. Th17 cells-induced diabetes is inhibited by adding anti-IFNγ antibodies, but not anti-IL-17 antibodies in a mouse diabetes model [45]. Thus, it is suggested that nonclassic Th1 cells are more important than Th17 cells per se or Th17/Th1 cells in the pathogenesis of autoimmune diseases.
On the other hand, the pathogenic role of nonclassic Th1 cells in patients with RA remains to be elucidated. In 2014, Miao et al. analyzed the association of circulating Th17 and Th1 cells expressing CD161 with disease activity in RA patients with disease duration of about 4 years under treatment with corticosteroids, disease-modifying anti-rheumatic drugs (DMARDs), or TNF inhibitors [46]. The percentages of CD161+Th17 and CD161+Th1 cells, but not CD161+Th17/Th1 cells, reflected the degree of RA activity. The findings of this study are interesting; however, the various medications possibly have an impact on the findings. Thus, the roles of these cells in the pathogenesis of RA need to be studied using peripheral blood from untreated and early-onset RA patients.
The previous studies of "Th1 cells" in human diseases may need to be evaluated again because "Th1 cells" were divided into classic Th1 cells and nonclassic Th1 [18][19][20][21]. Detection of Th17/Th1 cells [30], CD161 as a marker of human Th17 cells [31], and CD161+Th1 cells as Th17-derived Th1 cells [18] were reported in 2007, 2008, and 2011, respectively. For example, it was reported in 2008 that Th1 cells but not Th17 cells play an important role in the pathogenesis of RA using samples from RA patients whose average disease duration was 13 years [47]. In this study, Th1, Th17, and Th17/Th1 were detected using only the production of cytokines IFNγ, IL-17, and both, respectively, without the detection of CD161. In addition, CD161 plays various roles as mentioned above, except for a role as a marker of human Th17 cells and nonclassic Th1 cells [38,39]. Thus, it is possible that "Th1 cells" included both classic Th1 cells and IL-17-derived nonclassic Th1 cells expressing CD161 in previous studies.
In 2016, we reported elevated ratios of CD161+Th1 cells (i.e., Th17 cell-derived Th1 cells to CD161+Th17 cells) in the peripheral blood of untreated and early-onset RA patients [48]. Recently, it has been reported that Th17 cells can shift to Th1 cells as mentioned above. However, it remained to be elucidated whether this shift occurs in the early phase of RA. In the study, we tried to identify Th17 cells, Th1 cells, and Th17 cell-derived Th1 cells (CD161+Th1 cells) in the peripheral blood of untreated and early-onset RA patients. We identified IL-17(+)IFN-γ(-)CD4(+) T cells as Th17 cells and IL-17(-) IFN-γ(+)CD4(+) T cells as Th1 cells, detecting CD161 on the cell membrane. We also evaluated the effect of methotrexate (MTX) on the ratio of Th17 cells in early-onset RA patients. The ratio of CD161+Th1 cells (i.e., Th17 cell-derived Th1 cells to CD161+Th17 cells) was elevated in the peripheral blood of early-onset RA patients. In addition, MTX reduced the ratio of Th17 cells but not Th1 cells or Th17/Th1 cells. These findings suggest that Th17 cells shift to Th1 cells even in the early phase of RA, and that Th17 cells play important roles in the early phase of RA. In addition, these findings suggest that anti-IL-17 antibodies should be administered to patients with RA in the early phase.

Th17-Producing IFNγ (IFNγ+ Th17)
The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artifacts. In 2012, Maecker et al. reported the standardization of cytometry assays and summarized the steps that are required for the Human Immunology Project [15]. In the standardization, the definition of particular subsets of immune cells is conducted using only cell-surface markers without measuring expressed cytokines or CD161.
In 2016, we reported that the ratio of circulating IFNγ+ Th17 cells in memory Th cells is inversely correlated with the titer of anti-CCP antibodies (ACPA) in untreated and early-onset RA patients using flow cytometry methods of the Human Immunology Project [49]. In the same study, we validated the methods of the Human Immunology Project using the cell-surface marker by measuring the actual expressions of IL-17 and IFNγ. In addition, we also evaluated CD161 as a marker of human Th17 cells, measuring the expression of CD161 in human Th17 cells [49].
We tried to identify Th17 cells, IL-17+Th17 cells, and IFNγ+Th17 cells in the peripheral blood of untreated and early-onset RA patients using the method of the Human Immunology Project [49]. Our findings validated the method and the expression of CD161. The ratio of IFNγ+Th17 cells in memory T cells was inversely correlated to the titers of anti-CCP antibodies. It has been reported that cell populations in synovial tissues may shift inversely to those in peripheral blood in RA [13]. Thus, we speculated that IFNγ+Th17 cells are infiltrated in synovial tissue with inflammation in RA patients with high titers of ACPA with decreased peripheral ratio of IFNγ+Th17. These findings suggest that anti-IL-17 antibodies should be administered to patients with early phase RA-especially those with high titers of CCP antibodies.

Possible Implications for Novel Therapies
Anti-IL-17 antibodies should be administered to early phase RA patients because Th17 cells shift to pathogenic Th17/Th1 or nonclassic Th1, even in the early phase of RA as mentioned above [48,49]. It is speculated that IL-17 is important in the initiation phase of RA, because it has been reported that serum level of IL-17 is higher before the onset of RA than afterward [12]. In phase II trials, anti-IL-17 antibodies, secukinumab or ixekizumab, showed an effect on disease activity of biologics-naïve RA patients or RA patients with an insufficient effect of TNF inhibitor or MTX [50][51][52][53][54][55]. However, phase III trials were closed early because there was no incremental benefit of IL-17 inhibition over other agents currently approved for use in patients who failed TNF inhibitors [56]. We expect that future clinical trials will be performed including only untreated and early-onset RA patients, although this may be difficult to perform.
Interestingly, in 2016, two groups reported the efficacy and safety of monoclonal antibodies targeting the IL-17 pathway for RA using a meta-analysis of randomized controlled clinical trials [57,58]. In addition, in 2017, using meta-analysis, Lee et al. reported associations between circulating IL-17 levels and RA and between IL-17 gene polymorphisms and disease susceptibility [59]. Their meta-analysis revealed significantly higher circulating IL-17 levels in patients with RA, and found evidence of associations between the IL-17A rs2275913, IL-17F rs763780, and IL-17A rs3819024 polymorphisms and pathogenesis of RA.
Interfering with IL-1β, TNFα, IL-23, and IL-12 is important to reduce Th17 cell differentiation into pathogenic Th17/Th1 cells or nonclassic Th1 cells [19,20]. In particular, TNFα is produced by Th17 cells per se. Thus, Th17 cells differentiate to nonclassic Th1 in an autocrine or a paracrine manner [9]. It is speculated that the TNF inhibitors used worldwide reduce the shift of Th17 cells to nonclassic Th1 cells as well as the direct inhibition of TNFα.
In vitro, TNFα and IL-17 show additive or synergistic effects in promoting the production of IL-6, IL-8, and granulocyte-colony stimulating factor (G-CSF), as well as matrix metalloproteinases (MMPs) [60]. Thus, inhibition of both TNFα and IL-17 is potentially attractive for the treatment of RA. Recently, a single antibody (CrossMab 2+2 ) combined at the site of Fc binding both IL-17 and TNFα was reported as a therapeutic opportunity in RA using human fibroblast-like synoviocytes (FLS) in vitro and arthritic mice in vivo [60,61]. In addition, another single antibody (ABT-122) binding both IL-17 and TNFα at the site of Fab was reported showing effectiveness and safety in RA therapy [62]. In addition, based on the marketed anti-TNF antibody adalimumab, Silacci et al. generated the bispecific TNF/IL-17-binding FynomAb COVA322 [60]. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. COVA322 is currently being tested in a Phase 1b/2a study in psoriasis [63]. These antibodies may reduce the shift of Th17 cells to nonclassic Th1 cells.
It is important to select RA patients with highly bioactive IL-17 to obtain a sufficient effect of the therapy using anti-IL-17 antibody. Ndongo-Thiam and Miossec constructed a functional bioassay system to measure circulating bioactive IL-17 [64]. Using the system, the IL-17 pro-inflammatory dependent level (IPDL) corresponding to bioactive IL-17 was measured as an anti-IL-17 antibody-inhibition level of IL-8 production from human umbilical vein endothelial cells (HUVEC). The levels were significantly higher in patients with RA versus healthy donors and in destructive versus non-destructive RA, with a positive correlation between IPDL and Larsen score [64]. Thus, this functional bioassay could be used for the selection of RA patients more likely to respond to anti-IL-17 antibody therapy.
Ustekizumab binds p40 (a common molecule of IL-12 and IL-23), and shows the effect of the therapy for psoriasis and psoriatic arthritis [65]. Recently, it has been reported that ustekizumab did not affect the disease activity of RA [66]. However, it is speculated that ustekizumab reduces the shift of Th17 cells to nonclassic Th1 cells.

Conclusions
Recent studies (including ours) show that the plasticity of Th17 plays important roles in the pathogenesis of RA. However, many issues with the plasticity of Th17 remain to be addressed. Resolution of these issues will provide more strategies for novel therapies.


text:
Interleukin-15 stimulates natural killer cell-mediated killing of both human pancreatic cancer and stellate cells

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in Western countries with a 5-year survival rate below 5%. One of the hallmarks of this cancer is the strong desmoplastic reaction within the tumor microenvironment (TME), orchestrated by activated pancreatic stellate cells (PSC). This results in a functional and mechanical shield which causes resistance to conventional therapies. Aiming to overcome this resistance by tackling the stromal shield, we assessed for the first time the capacity of IL-15 stimulated natural killer (NK) cells to kill PSC and pancreatic cancer cells (PCC). The potency of IL-15 to promote NK cell-mediated killing was evaluated phenotypically and functionally. In addition, NK cell and immune checkpoint ligands on PSC were charted. We demonstrate that IL-15 activated NK cells kill both PCC and PSC lines (range 9-35% and 20-50%, respectively) in a contact-dependent manner and significantly higher as compared to resting NK cells. Improved killing of these pancreatic cell lines is, at least partly, dependent on IL-15 induced upregulation of TIM-3 and NKG2D. Furthermore, we confirm significant killing of primary PSC by IL-15 activated NK cells in an ex vivo autologous system. Screening for potential targets for immunotherapeutic strategies, we demonstrate surface expression of both inhibitory (PD-L1, PD-L2) and activating (MICA/B, ULBPs and Galectin-9) ligands on primary PSC. These data underscore the therapeutic potential of IL-15 to promote NK cell-mediated cytotoxicity as a treatment of pancreatic cancer and provide promising future targets to tackle remaining PSC.

A hallmark of PDAC is the strong desmoplastic reaction which occurs in the tumor microenvironment (TME) resulting in a dense fibrotic/desmoplastic stroma that surrounds the pancreatic cancer cells (PCC) [11][12][13]. This stroma, which can cover more than 50% of the tumor, acts as a mechanical and functional shield causing a diminished delivery of systemically administered anticancer agents due to a high intratumoral pressure and low microvascular density [11][12][13][14][15]. The main orchestrators of this phenomenon are the activated pancreatic stellate cells (PSC). These myofibroblast-like cells enhance the development, progression and invasion of PDAC through their extensive crosstalk with the PCC, resulting in reciprocal stimulation and therapy resistance. It is strongly believed that (new) treatment options will only be able to exert their full potential provided that this shield is breached [11-14, 16, 17].
In several cancer types immunotherapy has prevailed where more conventional therapies have failed. In 2015, the Cancer Immunotherapy Trials Network (CITN) ranked the immune checkpoint inhibitors targeting programmed death (PD-)1 or its ligand PD-L1 as the number 1 on the priority list for immunotherapeutic agents [18]. These blocking antibodies have taken a leap forward in the struggle against several cancers, including melanoma and lung cancer [19,20]. Unfortunately, despite the fact that PD-1 and PD-L1 are expressed in PDAC, anti-PD-1 therapy achieved only little effect [7,21].
Interleukin (IL)-15, ranked third on the CITN priority list [18], is a highly attractive cytokine for immune cell stimulation because it can increase the proliferation and persistence of natural killer (NK) and T cells but does not -in contrast to IL-2 -stimulate immune-suppressing regulatory T cells (Tregs) [22]. The majority of (novel) immunotherapeutic strategies focuses on activating T cells. However, innate immune cells -especially NK cells -are less often suppressed by current chemotherapeutic treatments and are demonstrated attractive effectors in immunotherapeutic strategies [23,24], since one of their main tasks is to target cancer cells [25]. In PDAC specifically, it has been shown that higher absolute levels of NK cells in circulation are associated with better survival [26]. Along the same lines, NKG2D -one of the major activating receptors of NK cells -is shown to be reduced on NK cells in PDAC patients [27]. Here, we are the first to look into the therapeutic potential of IL-15 stimulated NK cells in PDAC. The aim of this study was to evaluate the potency and mechanism of IL-15 to promote NK cell-mediated killing of PCC and PSC, the latter in order to breach the stromal shield surrounding the tumor rendering the tumor more susceptible for anticancer agents. Additionally, we screened PSC for a range of prototypic NK cell and highly attractive immune checkpoint ligands with a view to grow our knowledge on novel therapeutic targets.

IL-15 potentiates NK cells from healthy donors to kill PCC and PSC lines
Depicted in Figure 1, purified unstimulated NK cells from healthy donors are capable of killing 2 out of 3 PCC lines (Mia-Paca-2 and BxPC-3; no killing of Capan-2) and all 3 PSC lines (hPSC128-S/V, RLT-PSC and hPSC21-S/T). Stimulation of NK cells with IL-15 clearly enhances NK cell-mediated cytotoxicity for all PCC and PSC lines tested with a statistically significant increase (range 1.3 to 5.3-fold) for all cocultures in the NK cell:target cell ratio 5:1. The dose-response analysis reveals that the 1:1 ratio has a lower amount of killing, whereas there is no general difference in killing between the ratios 10:1 and 5:1.

Direct cell-cell contact between NK cell and PCC/PSC is necessary for killing
A transwell assay was performed to investigate the need for direct cell-to-cell contact between the (un) stimulated NK cells and the PCC or PSC in order to get killing. A statistically significant difference was observed for all cell lines between the transwell setting and the control setting where effector and target cells were in direct contact ( Figure 2). Our data show that unstimulated as well as stimulated NK cells need direct cell-to-cell contact with the target cells, indicating that membrane factors play a major role to effectuate a cytotoxic effect of NK cells towards PCC and PSC cells. No killing was observed when effector and target cells were separated by a transwell.

IL-15 results in upregulation of NKG2D and TIM-3 on NK cells from healthy donors
To unravel the mechanism via which IL-15 potentiates NK cells to kill PCC and PSC, the level of expression of a range of regulating molecules on both unstimulated and IL-15 stimulated NK cells was quantified. The receptors NKp46, NKG2D, DNAM-1, TIM-3 and 2B4 were all clearly expressed on unstimulated NK cells with ΔMFI levels above 25. PD-1, LAG-3, FasL and TRAIL were less expressed (ΔMFI levels ranging from 2 to 12). Only NKG2D (1.8-fold increase) and TIM-3 (2.4-fold increase) were statistically significantly increased after IL-15 stimulation ( Figure 3A). Looking at the percentage of cells expressing the receptors, 70 to 91% of the unstimulated NK cells were positive for NKp46, DNAM-1 and 2B4; 25 to 41% were positive for NKG2D, TIM-3 and FasL; while PD-1, LAG-3 and TRAIL presence ranged from 6% to 25%. Similar to the MFI data, IL-15 induced a significant increase of NKG2D (1.4-fold) and TIM-3 (1.5-fold) positive NK cells ( Figure 3B).

NKG2D is partially responsible for the augmented killing of PCC and PSC by IL-15 stimulated NK cells
We blocked NKG2D and TIM-3 using monoclonal neutralizing antibodies to investigate whether these proteins play a role in the higher killing rates after IL-15 stimulation. Blocking of NKG2D resulted in a statistically significant decrease of killing by IL-15 stimulated NK cells in all PCC and PSC ( Figure 4). Remarkably TIM-3 neutralization resulted in a significant decrease in cytotoxicity for 2 PCC lines, Mia-Paca-2 and Capan-2. Within our PSC line panel, no significant difference in cytotoxicity was observed after blocking TIM-3 on IL-15 stimulated NK cells. Our data indicate that NKG2D is partially responsible for the augmented killing of PSC and PCC by IL-15 stimulated NK cells, while for TIM-3 this is only the case for PCC.

IL-15 stimulated NK cells are capable of killing primary PSC in an autologous setting
As shown in Figure 5., we investigated the capacity of IL-15 stimulated NK cells to kill PSC in an autologous setting ( Figure 5A). In 4 out of 5 PDAC patient samples tested, stimulation of NK cells with IL-15 resulted in a statistically significant increased capacity of killing of autologous PSC derived from tumor tissue, ranging from 23.8% to 52.9% killing after stimulation (2.7-to 6.9-fold increase; p < 0.05) at an E:T ratio of 5:1. Interestingly, these killing rates are similar compared with killing of PSC cell lines in an allogeneic setting ( Figure 1, ranging from 18.3% to 50% for ratio 5:1). For patient 3, 10.9% of PSC were killed by unstimulated autologous NK cells with no further increase following IL-15 stimulation.
In analogy with healthy donor NK cells, the expression of NKG2D and TIM-3 on unstimulated versus IL-15 stimulated patient NK cells is significantly increased by 1.7-and 2.8-fold respectively (p<0.05 for both markers) ( Figure 5B). Similarly, the percentage of PDAC patient NK cells positive for NKG2D and TIM-3 is significantly increased following IL-15 stimulation from 16.4% to 23.6% and from 41.2% to 57.9% positive NK cells, respectively ( Figure 5C). NKG2D expression on patient NK cells was lower than on NK cells of healthy donors, also after IL-15 stimulation, whereas TIM-3 expression was comparable. A similar trend is seen for the percentage of NK cells expressing these two receptors ( Figure 5B and 5C).
PSC derived from the PDAC tissue samples were evaluated for the NKG2D ligands MICA/B and different ULBPs, the TIM-3 ligand Galectin-9 and the PD-1 ligands PD-L1 and PD-L2. These ligands were all present on PSC, with ΔMFI ranging from 56 to 403 ( Figure 5D) and the percentage of positive cells from 14 to 61% ( Figure  5E). No correlation was found between the amount of expression of the ligands and the amount of killing of the PSC (data not shown).

DISCUSSION
In this study we looked into the potential of (IL-15 stimulated) NK cells to kill PSC. Depletion of PSC and thus the stromal compartment has proven successful since the addition of the chemotherapeutic agent nab-Paclitaxel, the albumin-bound form of paclitaxel which accumulates in the tumor cells and stroma [28], results in better survival than chemotherapy alone [9,29]. Although there is some evidence that depletion of the stroma might also cause faster tumor progression [30,31], several studies showed that stromal depletion combined with immunomodulation had better outcomes than immunomodulation alone [30,32]. Currently, several other strategies are being investigated in which stromal depletion is pursued, however none have explored the potential of NK cells [11,33,34]. To our knowledge, we are the first to describe NK cell-mediated killing of PSC.
In addition, we show that NK cells are capable of killing the PCC. Our results are in concordance with those of others who showed that NK cells from PDAC patients are capable of killing PCC lines, other than the ones used in this study [27,35]. Although IL-15 has recently been acknowledged as a versatile molecule with great potential in cancer immunotherapy and NK cell stimulation [22,[36][37][38], none of these PCC studies evaluated IL-15 activated NK cells. IL-15 has already proven to have a beneficial effect in PDAC since Yoshida et al. showed that IL-15 producing PDAC cells induced a tumor reducing effect via NK cells [39] and Jing et al. showed that human umbilical cord blood-derived mesenchymal stem cells producing IL-15, eradicate pancreatic tumors in mice [40]. Furthermore, it had been demonstrated that NK-92, an IL-2 dependent human NK cell line, is able to kill several PCC lines. Here, a similar amount of killing for the CAPAN-2 cell line was observed but the Mia-Paca cell line was killed at least 40% less than with IL-15 stimulated NK cells evaluated in this study, stressing the potential of IL-15 [41]. IL-15 has proven safe for administration in humans in a first clinical trial [42]. These results mark an important milestone for IL-15 as a highly promising anticancer drug that has beneficial properties compared to IL-2. Treatment with high doses (due to the very short half-life) of IL-2 causes vascular leak syndrome, pulmonary edema, hypotension and heart toxicities, as well as expansion of the immunosuppressive Tregs [43]. IL-15 has a lower toxicity profile and does not stimulate expansion of Tregs. Moreover, a new stabilized IL-15 product, the superagonist ALT-803, has shown great potential in cancer [44,45]. Currently several clinical trials are investigating IL-15 for cancer treatment of which one investigates ALT-803 in combination with gemcitabine and nab-Paclitaxel in PDAC (NCT02559674). In the phase IB/II trial, the maximum tolerated dose, safety profile and overall survival will be tested. Our data show that IL-15 has the potential to play an important role in the treatment of PDAC by stimulating NK cells to target both tumor cells and the surrounding desmoplastic barrier.
A hurdle to overcome for NK cell-mediated immunotherapy in PDAC is that NK cells have decreased activity in PDAC patients [46]. We observed a lower expression of the activating receptor NKG2D on NK cells from the PDAC patients as compared to healthy control NK cells. These results are in concordance with data presented by others [27,47]. Our results also show that NKG2D is partially responsible for the mechanism via which PSC and PCC are killed by IL-15 stimulated NK cells. As demonstrated by Horng et al. in mice [48] in a non-PDAC context, IL-15 is capable of increasing NKG2D expression on patient NK cells. Furthermore, we observed an upregulation of TIM-3 on NK cells after IL-15 stimulation. This is in accordance with previous findings, also in a non-PDAC context [49]. Concerning the role of TIM-3 on NK cells, contradictory results have been published [50]. On the one hand, Ndhlovu et al. showed that cytotoxic capacity and IFN-γ production were associated with NK cells expressing the highest TIM-3 levels [49] and in accordance with our data, Gleason et al. demonstrated that TIM-3 was upregulated after NK cell activation and promoted IFN-γ production in response to its ligand Galectin-9 [51]. These results suggest that TIM-3 is a marker of fully functional and mature NK cells and has an activating role, in contrast with data of Ndhlovu et al., who demonstrated that NK cell-mediated cytotoxicity is suppressed when TIM-3 is activated by crosslinking antibodies or after encounter with target cells expressing Galectin-9 [49]. Accordingly, it has been shown that TIM-3 blockade improved NK cell-mediated cytotoxicity in human lung adenocarcinoma and advanced melanoma [52,53]. We observed reduced killing of PCC after TIM-3 blockade which is in contradiction with the results of the latter. These data underscore the ambiguous role of TIM-3 on NK cell function in cancer and warrants further investigation.
As for the effect of IL-15 on the activating NK cell receptors NKp46, DNAM-1 and 2B4 (i.e. no changes), our observations are similar to the ones found by others [54,55]. For the expression of FasL, we observed slightly higher expression than Feng et al., while they show strong upregulation of TRAIL which we did not observe [54]. These differences might be related to their longer IL-15 stimulation period of 10-12 days. Regarding LAG-3, we observed no upregulation of the protein. Although one study observed an upregulation in LAG-3 and PD-1 mRNA after IL-15 stimulation of NK cells, we are the first to describe the effect on LAG-3 on protein level [56].
In order to gain mechanistic insight in the susceptibility of PSC for NK cell-mediated killing, we screened PSC from the PDAC patients for NK cell ligands.    [57]. Regarding the expression of Galectin-9, we are the first to describe the expression of this TIM-3 ligand on PSC. Moreover, we assessed the expression of PD-L1 and PD-L2, since it has already been shown that blockade of PD-L1 combined with inhibition of IL-6 produced by PSC has beneficial effects on PDAC in murine models [58]. We demonstrate both PD-L1 and PD-L2 surface expression on primary PSC using flow cytometry, which is in concordance with immunohistochemistry data shown by Nomi et al [59]. The expression of these ligands can be of interest as future points of action for novel immunotherapies that also aim to target the cancer cells as well as the shield made by the PSC.

Ethics statement
This study was approved by the local Ethics Committee of the University of Antwerp (Antwerp, Belgium) under the reference number 14/47/480. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood samples from patients with pancreatic ductal adenocarcinoma (PDAC) using a Ficoll density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). From the same patients, tissue was obtained from the resected tumor after Whipple procedure. For healthy controls, PBMC were isolated from adult volunteer whole blood donations supplied by the Red Cross Flanders Blood Service (Mechelen, Belgium). Selection of the donors and collection of blood was performed according to Belgian law and Belgian Red Cross policy.  [61] are cultured in DMEM-F12 (1:1) supplemented with 10% FBS and 2mM L-Glutamine. Cell lines were split twice a week and incubated at 37°C with 5% CO 2 .

Human (primary) cell lines
Primary PSC were cultured from human PDAC tissue samples using an outgrowth method [62]. Briefly, PDAC tissue samples were put in a sterile petri dish and cut in small pieces of 2-3 mm 3 using a scalpel. Next, the tissue pieces were transferred to a 75 cm 2 culture flask and incubated in DMEM-F12 supplemented with 10% FBS, 2mM L-Glutamine, 500 U/ml penicillin and 500 μg streptomycin. Culture medium was changed twice a week. After an average of 3 weeks, PSC spontaneously grew out of the tissue pieces. Cells were passaged using trypsin-EDTA and incubated at 37°C and 5% CO 2 . Characterization of the primary PSC was performed by checking expression of the following markers [63]: α-smooth muscle actin (α-SMA), glial fibrillary acidic protein (GFAP), Vimentin and Desmin using an immunohistochemistry (IHC) staining protocol as described before with minor modifications [60].

NK cell isolation and stimulation
Cryopreserved PBMC where thawed and incubated overnight at 37°C and 5% CO 2 in complete medium (RPMI 1640 supplemented with 10% FBS, 2mM L-Glutamine, 100 U/ml penicillin, 100 µg streptomycin and sodium-pyruvate). Subsequently, NK cells were isolated using negative magnetic activated cell sorting (MACS), according to the manufacturer's protocol (Miltenyi Biotec). After isolation, purity of the NK cells -measured by flow cytometric immunophenotyping the cells with CD3-FITC (Immunotools) and CD56-PE (BD Biosciences) -was above 90%. NK cells were split in 2 equal portions; one to stimulate with 10 ng/mL recombinant human IL-15, while the other portion was left untreated. Both conditions were incubated overnight at 37°C and 5% CO 2 .

NK cell-mediated cytotoxicity assays
In order to measure the cytotoxic capacity of (un) stimulated peripheral blood NK cells towards PCC and PSC, a flow cytometric assay was used as described before with minor adjustments [64][65][66]. Briefly, PCC and PSC were labelled with the green fluorescent membrane dye PKH-67 (Sigma Aldrich) according to manufacturer's protocol and served as 'target cells'. PKH-67-positive target cells were put in coculture with (un)stimulated effector NK cells at three different effector:target (E:T) ratio's: 10:1, 5:1 and 1:1. In the ex vivo autologous experiments, only the 5:1 ratio was used. Tumor cells incubated without NK cells served as controls.
The necessity of direct cell-cell contact between target and effector cells was investigated by using a transwell assay which prevented direct contact. PKH-67 labelled target cells were put in the bottom compartment of a 96-well transwell plate (HTS Transwell ® -96 Well, Pore size 0.4μm, Corning) and (un)stimulated target cells were added in the top compartment at an E:T ratio of 5:1. Cocultures of effector and target cells with www.impactjournals.com/oncotarget direct cell-cell contact served as positive controls while cultures of tumor cells without effector cells served as negative controls.
In a specific set of experiments, the involvement of NKG2D and TIM-3 in NK cell-mediated killing of PCC and PSC was measured by a 2h preincubation of (un) stimulated NK cells with 20 mg/ml anti-NKG2D (R&D Systems), anti-TIM-3 (eBioscience) or corresponding isotype control antibodies (Jackson Laboratories) prior to coculture at an E:T ratio of 5:1.
In all experiments, cocultures incubated 4h after which the supernatant was discarded and cells were acquired on the FACS Aria II (BD Biosciences) after Annexin V-APC (BD Biosciences) and propidium iodide (PI) (Thermo Fisher Scientific) staining for quantification of the target cell viability. Target cell killing by NK cells was calculated by the following formula:

Phenotyping of target and effector cells
The immunophenotype of purified (un)stimulated NK cells was evaluated using PE-conjugated antibodies (BD Biosciences) and flow cytometry for the following surface markers that might have a stimulating or inhibiting effect on NK cell functions: NKG2D, DNAM-1, PD-1, TIM-3, LAG-3, NKp46, FasL, TRAIL and 2B4. In addition, corresponding ligands on the PCC and (primary) PSC were measured using PE-conjugated antibodies and flow cytometry for the following markers: MICA/B, ULBP1, ULBP2,5,6, ULBP3, ULBP4 (NKG2D ligands; antibodies all from R&D Systems), Galectin-9 (TIM-3 ligand; antibody from BD Biosciences), PD-L1 and PD-L2 (PD-1 ligands; antibodies both from BD Biosciences). Corresponding isotype controls were included for all samples and served as negative controls. All samples were measured on a FACSCan flow cytometer (BD Biosciences).

Data analysis
All flow cytometry data were analyzed using FlowJo v10.1. GraphPad Prism 7 was used for data comparison and artwork. Statistical analyses were performed with IBM SPSS Statistics 23. Paired-wise non-parametric Wilcoxon signed ranks testing was performed to compare means. Correlations were investigated using the Spearman's correlation coefficient. Differences were predefined to be considered as statistically significant when p < 0.05.

CONCLUSION
We show that NK cells are capable of killing both PCC and PSC and this significantly better after stimulation with IL-15. These results support the importance and value of an IL-15 and NK cell orientated approach to treat PDAC. NKG2D is partially responsible for this effect, as well as TIM-3 to a lesser degree. Our observations regarding the expression and presence of several NK cell ligands on primary PSC reveal possible future targets to tackle remaining PSC. Further research to investigate the in vivo potential of this new immunotherapeutic approach in PDAC will be performed in the near future.