Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro and in vivo

Background

Hypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.

Methodology/Principal Findings

Here we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.

Conclusions/Significance

These findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.     

Antitumor effect of recombinant tumor necrosis factor-α against murine sarcomas at visceral sites: tumor size influences the response to therapy

Summary We examined the antitumor efficacy of rTNF- administration on established tumor at two visceral sites, lungs and liver. Treatment of B6 mice harboring multiple (>100 foci of 0.5 mm diameter) 10-day pulmonary macrometastases from the MCA-106 sarcoma, with dosages of rTNF- (5–10 g, single dose i. v.) that caused hemorrhagic necrosis and regression of a 6 mm MCA-106 s. c. tumor, had no impact on the number (or size) of lung nodules. Similarly, rTNF- failed to show an antitumor effect in B6 mice with advanced day 8 or 10 multiple (>100 foci of 0.5 mm diameter) hepatic metastases at single i. v. doses up to 20 g, as measured by either enumeration of residual liver nodules or survival. B6 mice injected s. c. with MCA-106 sarcoma and treated with rTNF- as a single i. v. dose on day 0, 3, 5, or 7 experienced marked tumor regression only after the day 7 rTNF- injection, when the tumor had achieved a size of 5–6 mm in diameter. Since tumor size appeared important for rTNF- susceptibility in vivo, we next induced a single hepatic tumor of the MCA-106 sarcoma by the direct injection of cells into the left lobe of the liver and treated these mice at day 10 when the nodule had achieved a size of 5–6 mm in diameter. Increasing doses of rTNF- (up to 8 g) given as a single i. v. injection resulted in increasingly greater reductions in hepatic tumor as well as significant survival benefit of the treated mice. Sites of regressing hepatic tumor exhibited central necrosis accompanied by polymorphonuclear leukocytes and lymphocytes. Collectively, these results show that rTNF- administration can mediate a significant antitumor effect on visceral tumor and suggest that tumor size is an important factor in rTNF- susceptibility not only for tumors growing at s. c. sites but also for those established at visceral sites.Howard Hughes Medical Institute Research Scholar Abbreviations used: rTNF-, recombinant tumor necrosis factor-; B6 mice, C57BL/6 mice; MCA, 3-methylcholanthrene; HBSS, Hanks' balanced salt solution; LAK, lymphokine-activated killer; rIL-2, recombinant interleukin-2     

Antitumor effect of combination of murine recombinant interferon β, murine recombinant interferon γ and human recombinant interleukin-2 in MethA-bearing mice

Summary We have previously reported that the combination of murine recombinant interferon (Mu-rIFN) with murine recombinant interferon (Mu-rIFN) provided greater inhibition of tumor growth than did each one alone in MethA-bearing mice. In the present study the effect of addition of human recombinant interleukin-2 (Hu-rIL-2) to the combination of Mu-rIFN with Mu-rIFN on tumor growth in BALB/c mice bearing syngeneic MethA fibrosarcoma was examined. Low doses of Hu-rIL-2 (5 × 10³ U or 5 × 10⁴ U at 3-day intervals) showed no antitumor activity, while a high dose of Hu-rIL-2 (5 × 10⁵ U) showed profound growth inhibition. The administration of IL-2 (ranging between 5 × 10³ U and 5 × 10⁵ U) in addition to the combination of IFN and IFN showed more augmented antitumor effects in a dose-dependent manner. Furthermore, the simultaneous administration of IL-2, IFN and IFN had more effective therapeutic activity, compared with the sequential administration of interferons and IL-2. These findings indicated that IL-2 in combination with IFN and was effective for cancer treatment.     

Enhanced sialylation and in vivo efficacy of recombinant human α-galactosidase through in vitro glycosylation

Human α-galactosidase A (GLA) has been used in enzyme replacement therapy for patients with Fabry disease. We expressed recombinant GLA from Chinese hamster ovary cells with very high productivity. When compared to an approved GLA (agalsidase beta), its size and charge were found to be smaller and more neutral. These differences resulted from the lack of terminal sialic acids playing essential roles in the serum half-life and proper tissue targeting. Because a simple sialylation reaction was not enough to increase the sialic acid content, a combined reaction using galactosyltransferase, sialyltransferase, and their sugar substrates at the same time was developed and optimized to reduce the incubation time. The product generated by this reaction had nearly the same size, isoelectric points, and sialic acid content as agalsidase beta. Furthermore, it had better in vivo efficacy to degrade the accumulated globotriaosylceramide in target organs of Fabry mice compared to an unmodified version. [BMB Reports 2013; 46(3): 157-162]     

Combined effects of interleukin-1α and transforming growth factor-β1 on modulation of human cardiac fibroblast function

During cardiac remodeling, cardiac fibroblasts (CF) are influenced by increased levels of interleukin-1α (IL-1α) and transforming growth factor-β1 (TGFβ1). The present study investigated the interaction between these two important cytokines on function of human CF and their differentiation to myofibroblasts (CMF). CF were isolated from human atrial appendage and exposed to IL-1α and/or TGFβ1 (both 0.1 ng/ml). mRNA expression levels of selected genes were determined after 6–24 h by real-time RT-PCR, while protein levels were analyzed at 24–48 h by ELISA or western blot. Activation of canonical signaling pathways (NFκB, Smad3, p38 MAPK) was determined by western blotting. Differentiation to CMF was examined by collagen gel contraction assays. Exposure of CF to IL-1α alone enhanced levels of IL-6, IL-8, matrix metalloproteinase-3 (MMP3) and collagen III (COL3A1), but reduced the CMF markers α-smooth muscle actin (αSMA) and connective tissue growth factor (CTGF/CCN2). By contrast, TGFβ1 alone had minor effects on IL-6, IL-8 and MMP3 levels, but significantly increased levels of the CMF markers αSMA, CTGF, COL1A1 and COL3A1. Co-stimulation with both IL-1α and TGFβ1 increased MMP3 expression synergistically. Furthermore, while TGFβ1 had no effect on IL-1α-induced IL-6 or IL-8 levels, co-stimulation inhibited the TGFβ1-induced increase in αSMA and blocked the gel contraction caused by TGFβ1. Combining IL-1α and TGFβ1 had no apparent effect on their canonical signaling pathways. In conclusion, IL-1α and TGFβ1 act synergistically to stimulate MMP3 expression in CF. Moreover, IL-1α has a dominant inhibitory effect on the phenotypic switch of CF to CMF induced by TGFβ1.     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

Inhibitory effects of tamoxifen and tumor necrosis factor α on human glioblastoma cells

We reported previously that tumor necrosis factor α (TNFα) inhibited proliferation and invasiveness of human malignant glial cells. Because tamoxifen, an estrogen antagonist, has also been shown to inhibit growth of such cells, we hypothesized that a combination of tamoxifen and TNFα might be more effective than either reagent alone. TNFα (1–100 ng/ml) or tamoxifen (80 ng/ml-2 μg/ml) alone inhibited proliferation of a human glioblastoma cell line (WITG3) in a dose-dependent fashion; in combination, tamoxifen and TNFα yielded additive growth inhibition. Apoptotic cells characterized by nuclear fragmentation were detectable after 48 h of TNFα or tamoxifen exposure and were significantly increased by combination treatment. In non-neoplastic human astroglia and fibroblasts, proliferation was unaffected by tamoxifen, and enhanced by TNFα as previously reported. Staurosporine (2–50 nM), which has been reported to augment the effects of TNFα, was less effective than tamoxifen against WITG3 and, in addition, was markedly inhibitory to non-neoplastic glial cells. Binding studies yielded no evidence of WITG3 estrogen or progesterone receptors, nor of tamoxifen effects on TNFα receptors. Data suggest that TNFα and tamoxifen in combination display growth-regulatory properties, which (a) are more inhibitory to human glioblastoma cells than either agent alone, (b) do not affect non-neoplastic glia, (c) do not require either estrogen/ progesterone receptors or alteration of external TNFα receptors, and (d) may involve apoptosis.     

Effects of human interleukin-1 on natural killer cell activity: is fever a host defense mechanism for tumor killing?

Interleukin-1 (IL-1) represents a family of polypeptides with a wide range of biological activities. cDNA from two gene products has been cloned; there are probably more. The human IL-1 family plays an important role in the pathogenesis of many diseases and functions as a key mediator of host response to various infectious, inflammatory, neoplastic, and immunologic challenges. Recombinant mouse (pI 5) and recombinant human (pI 7) IL-1s are being used to confirm the multiple biological properties of IL-1s. Some IL-1 biological activities seem to be involved with mechanisms of host tumor killing. Incubating purified or recombinant human IL-1 with human peripheral blood mononuclear cells in the presence of IL-2 or interferon-alpha results in a synergistic enhancement of certain tumor cells. More recent results indicate that IL-1 exhibits direct cytotoxicity for tumor cells in vitro. The peripheral blood mononuclear cells of patients with tumors demonstrate decreased production of IL-1 when challenged with endotoxin and show a comparable decrease in natural killer activity; adding exogenous IL-1 reverses this defect in these patients. However, induction of hepatic acute-phase proteins such as serum amyloid A serves as a negative feedback since the amyloid protein suppresses natural killer activity. Moreover, natural killer cell activity in the presence of IL-1 or interferon-alpha is suppressed by incubating temperatures of 39 degrees C. This effect is not reversed by inhibitors of prostaglandin synthesis. IL-1 is clearly important to host defense against malignancy, but some aspects of IL-1 biology seem to exert a contrary influence.     

Combined anti-tumor effects of IFN-α and sorafenib on hepatocellular carcinoma in vitro and in vivo

Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide, and novel therapeutic strategies are urgently required to improve clinical outcome. Interferon-alpha (IFN-α) and sorafenib are widely used as anti-tumor agents against various malignancies. In this study, we investigated the combined effects of IFN-α and sorafenib against HCC. We demonstrated that the combination therapy synergistically suppressed HCC cellular viability, arrested cell cycle propagation and induced apoptosis in HCC cells. Further research revealed that IFN-α and sorafenib collaboratively regulated the expression levels of cell cycle-related proteins Cyclin A and Cyclin B as well as the pro-survival Bcl-2 family proteins Mcl-1, Bcl-2 and Bcl-X(L). Moreover, sorafenib inhibited IFN-α induced oncogenic signaling of STAT3, AKT and ERK but not the activation of the tumor suppressor STAT1. Xenograft experiments also confirmed the combined effects of IFN-α and sorafenib on tumor growth inhibition and apoptosis induction in vivo. In conclusion, these results provide rationale for the clinical application of IFN-α and sorafenib combination therapy in HCC treatment.     

Low pH inactivation for xenotropic gamma retrovirus in recombinant human TNF-α receptor immunoglobulin G and mechanism of inactivation

CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in CHO cells. Validation study for inactivation process is required. Murine xenotropic gamma retrovirus (X-MulV) is a model virus in validation study. In our previous study, optimum conditions for X-MulV inactivation were sifted. In this study, we performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of recombinant human TNF-α receptor immunoglobulin G fusion proteins (rhTNF-α) for injection. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-α were spiked with X-MulV and were inactivated at pH 3.60 ∼ 3.90, 25 ± 2 °C, and 0 ∼ 240 min, respectively. Samples incubated at the conditions for 15 ∼ 180 min were not inactivated effectively. For 4 h incubation, log₁₀ reductions were achieved 5.0 log₁₀. Biological activity of rhTNF-α incubated at pH 3.60, 25 °C for 4 h, which was assayed on murine L929 fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH 3.60, and it may be the mechanism of low pH inactivation.     

Inhibitory effects of tumor necrosis factor-α on cationic lipid-mediated gene delivery to airway cells in vitro

Cationic liposomes have been used successfully for DNA delivery to airway cells in vitro and are being tested in human clinical trials for their efficacy in cystic fibrosis transmembrane conductance regulator (CFTR) gene delivery in cystic fibrosis patients. While cationic liposomes are effective for transfection of airway cells in culture, they have not been effectively used for gene delivery to human airway cells in vivo. Several barriers in cystic fibrosis lungs, including increased amounts of mucus, phagocytic cell activity and cytokine-rich milieu caused by inflammation, may cause inhibition of gene transfection. As presented in this paper, we examined the effects of inflammatory cytokines on cationic lipid-mediated transfection of model airway cells. The results of these experiments indicate that tumor necrosis factor (TNF)-α dramatically inhibits Lipofectin-mediated transfection efficiency of H441 cells. Addition of anti-TNF-α neutralizing antibody results in recovery of efficiency. Results of temporal studies are consistent with the concept that TNF-α reduces transfection efficiency by a mechanism(s) other than or in addition to gene expression. These results are corroborated by fluorescence microscopic experiments which demonstrate that endocytosis of lipoplex is altered in the presence of TNF-α.     

In vivo tumorigenicity and in vitro sensitivity to tumor-necrosis-factorα mediated killing of c-Ha-ras-transformed cells

Summary Cellular subclones of high and low tumorigenicity obtained from a mouse c-Ha-ras-transformed clone, were examined for their sensitivity to tumornecrosis-factor (TNF)-mediated cytotoxicity. Cells of the highly tumorigenic subclones showed a significantly enhanced resistance to the cytotoxic effect of TNF plus cyclohexamide (CHI) as compared to cells of the lowtumorigenic subclones. The enhanced resistance to TNF+CHI was not due to a lower expression of TNF receptors on the cells. The c-Ha-ras-transfected cells were transformed and maintained in culture only (C cells). In vivo passage of cells of the initially low-tumorigenic c-Ha-ras subclones through the mouse significantly enhanced the tumorigenic potential of these CTC cells (culture/tumor/culture). In correlation with their enhanced tumorigenicity, the CTC cells were highly resistant to TNF-mediated cytotoxicity as compared to C cells of the same subclone. Furthermore, the involvement of TNF in determining the tumorigenic phenotype of the c-Ha-ras-transformed cells was demonstrated in a more direct manner. Cells of a c-Ha-ras-transformed low-tumorigenic, highly TNF-sensitive subclone were selected by repeated cycles of in vitro exposure to TNF. As a result, a stable, highly TNF-resistant population of cells emerged. These TNF-resistant cells caused more tumors in mice as compared to their original TNF-sensitive cells. These results show that the resistance to the cytotoxic effect of TNF plus cyclohexamide may be involved, at least partially, in the tumorigenic potential of c-Ha-ras-transformed cells and suggest a possible role for TNF in the enhancement of the tumorigenic potential of these cells in mice.     

In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells

Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA + βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA + βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.     

N⁴-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: cytotoxicity against human tumor cells, structure-activity relationship studies and investigation on the mechanism of action

N(4)-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene)hydrazinecarbothioamide) and its N(4)-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh, H2Ac4pFPh), N(4)-ortho-, -meta- and -para-chlorophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh), N(4)-ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh, H2Ac4pIPh) and N(4)-ortho-, -meta- and -para-nitrophenyl (H2Ac4oNO(2)Ph, H2Ac4mNO(2)Ph, H2Ac4pNO(2)Ph) derivatives were assayed for their cytotoxicity against human malignant breast (MCF-7) and glioma (T98G and U87) cells. The compounds were highly cytotoxic against the three cell lineages (IC(50): MCF-7, 52-0.16 nM; T98G, 140-1.0 nM; U87, 160-1.4 nM). All tested thiosemicarbazones were more cytotoxic than etoposide and did not present any haemolytic activity at up to 10(-5)M. The compounds were able to induce programmed cell death. H2Ac4pClPh partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization.     

Polyamine-containing etoposide derivatives as poisons of human type II topoisomerases: Differential effects on topoisomerase IIα and IIβ

Etoposide is an anticancer drug that acts by inducing topoisomerase II-mediated DNA cleavage. Despite its wide use, etoposide is associated with some very serious side-effects including the development of treatment-related acute myelogenous leukemias. Etoposide targets both human topoisomerase IIα and IIβ. However, the contributions of the two enzyme isoforms to the therapeutic vs. leukemogenic properties of the drug are unclear. In order to develop an etoposide-based drug with specificity for cancer cells that express an active polyamine transport system, the sugar moiety of the drug has been replaced with a polyamine tail. To analyze the effects of this substitution on the specificity of hybrid molecules toward the two enzyme isoforms, we analyzed the activity of a series of etoposide-polyamine hybrids toward human topoisomerase IIα and IIβ. All of the compounds displayed an ability to induce enzyme-mediated DNA cleavage that was comparable to or higher than that of etoposide. Relative to the parent drug, the hybrid compounds displayed substantially higher activity toward topoisomerase IIβ than IIα. Modeling studies suggest that the enhanced specificity may result from interactions with Gln778 in topoisomerase IIβ. The corresponding residue in the α isoform is a methionine.     

TRAIL(+) human plasmacytoid dendritic cells kill tumor cells in vitro: mechanisms of imiquimod- and IFN-α-mediated antitumor reactivity

Dendritic cells (DCs) not only exhibit the unique capacity to evoke primary immune responses, but may also acquire TLR-triggered cytotoxic activity. We and others have previously shown that TLR7/8- and TLR9-stimulated plasmacytoid DCs (pDCs) isolated from human peripheral blood express the effector molecule TRAIL. The exact mechanisms through which pDCs acquire and elicit their cytotoxic activity are still not clear. We now show that in the absence of costimulators, TRAIL induction on pDCs occurs with agonists to intracellular TLRs only and is accompanied by a phenotypic as well as functional maturation, as evidenced by a comparatively superior MLR stimulatory capacity. pDCs acquired TRAIL in an IFN-α/β-dependent fashion and, notably, TRAIL expression on pDCs could be induced by IFN-α stimulation alone. At a functional level, both TLR7/8- (imiquimod [IMQ]) and TLR9-stimulated (CpG2216) pDCs lysed Jurkat T cells in a TRAIL- and cell contact-dependent fashion. More importantly, IFN-α-activated pDCs acquired similar cytotoxic properties, independent of TLR stimulation and maturation. Both IMQ- and IFN-α-activated pDCs could also lyse certain melanoma cell lines in a TRAIL-dependent fashion. Interestingly, suboptimal doses of IMQ and IFN-α exhibited synergistic action, leading to optimal TRAIL expression and melanoma cell lysis by pDCs. Our data imply that tumor immunity in patients receiving adjuvant IMQ and/or IFN-α may involve the active participation of cytotoxic pDCs.