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Mechanism of NK cell activation induced by coculture with dendritic cells derived from peripheral blood monocytes
Abstract
Dendritic cells (DCs) have been regarded as one of the effective antigen-presenting cells, but the relationship between DCs and lymphocytes, in particular natural killer (NK) cells, remains unclear. In this study, we evaluated how DCs interact with both lymphocytes and NK cells using a coculture system. The number of lymphocytes increased significantly when cocultured with DCs (1.8-fold increase). In particular, the proliferation of NK cells was prominent. Furthermore, the coculture of DCs with lymphocytes induced a marked increase in IL-12 and IFN-gamma secretion. When contact between the DCs and lymphocytes was prevented, the secretion of both IL-12 and IFN-gamma was markedly reduced. IFN-gamma production was completely blocked by an anti-IL-12 antibody, indicating that IFN-gamma secretion was dependent on IL-12 secretion. The stimulating effect of the DCs on the proliferation of the lymphocytes was partially suppressed by anti-IL-12 antibodies, and was completely attenuated when cellular contact was prevented. Furthermore, the NK cell proliferation induced by coculture with DCs was significantly blocked by the inhibition of the interaction of either CD40-CD40L or CD28-B7 molecule. The coculture with DCs enhanced NK activity by 40%, and this was partially suppressed by anti-IL-12 antibodies and was completely blocked by the inhibition of cell-to-cell contact. These results indicate that the activation of NK cells by DCs is partially mediated by IL-12 secretion, and that direct contact between DCs and NK cells play a major role in this response.
... Yu et al. (24) demonstrated the capability of CD34 ϩ cell-derived DCs to significantly improve the tumor-directed cytotoxicity and IFN-␥ secretion of NK cells. In addition, it has been shown that the expansion, IFN-␥ production, and tumoricidal potential of NK cells are increased efficiently upon interaction with monocyte-derived DCs (25)(26)(27). Cross-talk is bidirectional because activated NK cells induce immature DC activation in terms of up-regulation of MHC and costimulatory molecules, resulting in an enhancement of the allostimulatory capacity of DCs (28,29). In addition, activated NK cells significantly increased the production of proinflammatory cytokines such as TNF-␣ and IL-12 by DCs. ...
... Recent studies demonstrated the ability of human monocytederived DCs to efficiently promote NK cell proliferation, IFN-␥secretion, and cytotoxicity (25)(26)(27). More recently, Munz et al. (44) illustrated that various myeloid DC subsets contribute to different properties of NK cells. ...
... These data suggest that membrane-associated determinants were essential for the augmentation of NK cell-mediated antitumor effects. This finding obtained with M-DC8 ϩ blood DCs is in accordance with several other reports demonstrating that the activation of NK cells by in vitro-differentiated DCs was critically dependent on cell-to-cell contact (23,24,26,28,29,46). However, the surface molecules, which are involved in DC-NK interaction, remain to be determined. ...
... Yu et al. (24) demonstrated the capability of CD34 ϩ cell-derived DCs to significantly improve the tumor-directed cytotoxicity and IFN-␥ secretion of NK cells. In addition, it has been shown that the expansion, IFN-␥ production, and tumoricidal potential of NK cells are increased efficiently upon interaction with monocyte-derived DCs (25)(26)(27). Cross-talk is bidirectional because activated NK cells induce immature DC activation in terms of up-regulation of MHC and costimulatory molecules, resulting in an enhancement of the allostimulatory capacity of DCs (28,29). In addition, activated NK cells significantly increased the production of proinflammatory cytokines such as TNF-␣ and IL-12 by DCs. ...
... Recent studies demonstrated the ability of human monocytederived DCs to efficiently promote NK cell proliferation, IFN-␥secretion, and cytotoxicity (25)(26)(27). More recently, Munz et al. (44) illustrated that various myeloid DC subsets contribute to different properties of NK cells. ...
... These data suggest that membrane-associated determinants were essential for the augmentation of NK cell-mediated antitumor effects. This finding obtained with M-DC8 ϩ blood DCs is in accordance with several other reports demonstrating that the activation of NK cells by in vitro-differentiated DCs was critically dependent on cell-to-cell contact (23,24,26,28,29,46). However, the surface molecules, which are involved in DC-NK interaction, remain to be determined. ...
Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity for DC-based cancer vaccination protocols. Novel findings reveal that besides their role as potent inducers of tumor-specific T cells, human DCs display additional antitumor effects. Most of these data were obtained with monocyte-derived DCs, whereas studies investigating native blood DCs are limited. In the present study, we analyze the tumoricidal capacity of M-DC8(+) DCs, which represent a major subpopulation of human blood DCs. We demonstrate that IFN-gamma-stimulated M-DC8(+) DCs lyse different tumor cell lines but not normal cells. In addition, we show that tumor cells markedly enhance the production of TNF-alpha by M-DC8(+) DCs via cell-to-cell contact and that this molecule essentially contributes to the killing activity of M-DC8(+) DCs. Furthermore, we illustrate the ability of M-DC8(+) DCs to promote proliferation, IFN-gamma production, and tumor-directed cytotoxicity of NK cells. The M-DC8(+) DC-mediated enhancement of the tumoricidal potential of NK cells is mainly dependent on cell-to-cell contact. These results reveal that, in addition to their crucial role in activating tumor-specific T cells, blood DCs exhibit direct tumor cell killing and enhance the tumoricidal activity of NK cells. These findings point to the pivotal role of DCs in triggering innate and adaptive immune responses against tumors.
... Studies focusing on human DCs revealed that monocyte-or stem cell-derived DCs enhance proliferation, IFN-g secretion and tumor-directed cytotoxicity of NK cells. [18][19][20][21] Crosstalk is bidirectional, as activated NK cells promote the maturation of DCs as well as their ability to secrete proinflammatory cytokines and to stimulate T-cell responses. 22,23 Although most of these data are based on DCs, which are generated after several days in the presence of various cytokines, studies investigating the interaction between native human DCs and NK cells are limited. ...
... In this context, it has been reported that monocyte-and stem cell-derived DCs promote proliferation, IFN-g secretion and tumor-directed cytotoxicity of NK cells. [18][19][20][21][31][32][33] Soluble factors such as IL-12, IL-15 and IL-18 as well as cell membrane-associated molecules contributed to NK cell activation mediated by these DCs. 19,21,31,32 However, the role of native human DCs as activators of NK cells is less well determined. ...
... [18][19][20][21][31][32][33] Soluble factors such as IL-12, IL-15 and IL-18 as well as cell membrane-associated molecules contributed to NK cell activation mediated by these DCs. 19,21,31,32 However, the role of native human DCs as activators of NK cells is less well determined. In this study, we demonstrated the ability of LPS-stimulated slanDCs, which represent a major subpopulation of human blood DCs, to efficiently activate resting NK cells. ...
Dendritic cells (DCs) display an extraordinary capacity to induce T-cell responses providing the opportunity of DC-based cancer vaccination strategies. Additional findings indicate that DCs may also play a crucial role for the activation of natural killer (NK) cells, which are important effectors in innate antitumor immunity. However, studies investigating the interaction between native human DCs and NK cells are limited. Recently, we defined 6-sulfo LacNAc (slan) DCs as a major subpopulation of myeloid human blood DCs, which represent principal producers of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin (IL)-12. Functional data revealed that slanDCs efficiently induce neoantigen-specific CD4+ T cells and activate tumor-reactive cytotoxic T cells. When evaluating the crosstalk between slanDCs and NK cells in this study, we found that lipopolysaccharide (LPS)-activated slanDCs efficiently enhance NK cell CD69 expression and interferon (IFN)-gamma secretion. NK cell-mediated tumor-directed cytotoxicity was significantly improved by slanDCs. NK cell activation induced by slanDCs was critically dependent on IL-12. When investigating the impact of NK cells on the immunostimulatory capacity of slanDCs, we observed that they promote DC maturation. In addition, NK cells strongly enhanced the secretion of immunomodulatory IL-12 and reduced the release of immunosuppressive IL-10 by slanDCs. IFN-gamma and cell-to-cell contact contributed to these effects. Furthermore, data revealed that DC-NK cell crosstalk improves slanDC-mediated differentiation of naïve CD4+ T lymphocytes into IFN-gamma-producing Th1 cells. In conclusion, we demonstrate a reciprocal activating interaction between slanDCs and NK cells, which may play a pivotal role in the regulation of antitumor immunity.
... Thus, activated DCs have been described to enhance CD69 expression, proliferation, IFN-γ secretion and cytotoxicity of NK cells via secretion of the monokine IL-12, TNF-α or type I IFNs 249 . For the induction of proliferation, also the contributions of B7 molecules on moDCs and CD28 on NK cells, trans-presented IL-15 as well as secreted IL-12 have been reported in the DC-stimulated NK proliferation 255,256 . In terms of cytokine induction, it has been demonstrated that the CX3CL1-CX3CR1 axis mediates the polarized secretion of IL-12 from DCs towards NK cells that in turn produce IFN-γ 257 . ...
... In terms of cytokine induction, it has been demonstrated that the CX3CL1-CX3CR1 axis mediates the polarized secretion of IL-12 from DCs towards NK cells that in turn produce IFN-γ 257 . Importantly, DCs also augmented the lytic capability of NK cells which was largely mediated by IL-12 and the CX3CL1-CX3CR1 axis 256,257 . ...
Seit der Entdeckung, dass verschiedene Immunzellen über die Fähigkeit verfügen, Tumorzellen zu erkennen und zu zerstören, wurden unterschiedliche Ansätze getestet, um unter anderem Natürliche Killerzellen (NK-Zellen) zur Therapie maligner Erkrankungen einzusetzen. So wurden mit NK-Zell-basierten Therapien wichtige Durchbrüche bei der Behandlung hämatologischer Tumore, doch nur geringe Erfolge in Patienten mit soliden Tumoren erzielt. Dass die Anzahl Tumor-infiltrierender NK-Zellen in vivo zu einer Verbesserung der Prognose führen kann, wurde jedoch für mehrere Krebsarten gezeigt. Wir vermuteten daher, dass es für das Ausbleiben relevanter therapeutischer Erfolge bei Patienten mit soliden Tumoren zwei Gründe geben könnte: Zunächst könnten die infundierten NK-Zellen evtl. nicht effizient in das Tumorgewebe eingewandert sein oder aber sie haben ihre Aktivität nach der Infusion in die Patienten rasch verloren. Das Ziel dieser Studie war es daher, die anti-Tumor-Aktivität adoptiv transferierter NK-Zellen zu verbessern, indem (1) die Anlockung von NK-Zellen in Tumore durch die Expression von chemotaktischen Zytokinen (Chemokinen) in Tumorzellen verbessert wird und (2) ein neuartiges Aktivierungsprotokoll etabliert wird, welches NK-Zellen mit lang anhaltender Aktivität generiert. Hierfür wurde zunächst ein Tiermodell entwickelt, um die in vivo-Relevanz unserer Studien zu belegen. Das Modell ermöglichte das Anwachsen humaner Luciferase-transduzierter Melanom-Zellen und NK-Zellen in immunsupprimierten Mäusen, in denen die Tumorgrößen durch Lumineszenzmessungen bestimmt werden konnten. Für unser erstes Ziel konnten wir weiterhin CX3CL1 als geeignetes Chemokin für die spezifische Anlockung zytotoxischer NK-Zellen identifizieren. CX3CL1-überexprimierende Melanom-Zelllinien lockten im Vergleich zu den jeweiligen Kontrollzellen NK-Zellen in vitro und in vivo im Xenograftmodell stärker an. Von großer Bedeutung ist, dass eine stärkere Kontrolle des Wachstums CX3CL1-positiver Tumore durch NK-Zellen in vivo gezeigt werden konnte. Für unser zweites Ziel testeten wir ein neuartiges Stimulierungsprotokoll. Cooper et al. haben in einer früheren Studie gezeigt, dass murine NK-Zellen, die mit Interleukin (IL)-12, IL-15 und IL-18 (IL-12/15/18) stimuliert worden waren, über lang anhaltende Reaktivität verfügten, weswegen wir dieses Aktivierungsprotokoll für humane NK-Zellen testeten. Als Referenz wurden IL-15-stimulierte NK-Zellen verwendet. Unsere Untersuchungen zeigten, dass IL-12/15/18-stimulierte NK-Zellen durch die verstärkte Expression der α-Kette des IL-2-Rezeptors sensitiver gegenüber IL-2 wurden. Demgemäß teilten sich die NK Zellen in vitro und in vivo in den Versuchstieren unter IL-2-Gabe schneller als die Kontrollzellen und lieferten größere Zellzahlen. Weiterhin reagierten IL-12/15/18-stimulierte NK-Zellen nach längerer in vitro-Kultivierung oder nach Re-Isolation aus Versuchstieren bei Restimulierung mit einer verstärkten Produktion von Interferon-γ (IFN-γ). Von großer Bedeutung ist weiterhin, dass die IL-12/15/18-Stimulierung eine stark verminderte Expression inhibitorischer Killer cell Immunoglobulin-like Receptors (KIR)-Rezeptoren bewirkte, deren niedrigere Expression auf NK-Zellen nach Stimulierung mit einer Zunahme der Reaktivität gegenüber Tumorzellen einherging, welche die zugehörigen inhibitorischen Liganden exprimierten. Zudem, verglichen mit IL-15-stimulierten Kontrollzellen, zerstörten IL-12/15/18-aktivierte NK-Zellen Tumorzellen in vitro stärker und verminderten das Tumorwachstum in vivo.
Zusammenfassend wird in dieser Arbeit beschrieben, dass sowohl die Anlockung von NK-Zellen durch die gezielte Expression von CX3CL1 in Tumorzellen, als auch die Aktivierung von NK-Zellen durch IL-12, IL-15 und IL-18 über ein großes therapeutisches Potential verfügen. In unserem prä-klinischen Mausmodell konnten wir eine verstärkte Anlockung von NK-Zellen in CX3CL1-exprimierende Tumore zeigen und für beide Ansätze eine verbesserte Tumorabstoßung durch NK-Zellen. Diese Daten verfügen damit über erhebliche klinische Relevanz und sollten in das Design klinischer Studien zur NK-Zell-basierten Tumorimmuntherapie einbezogen werden.
... The interaction between dendritic cells (DCs) and T cells has been well studied 14,16 -20 and recently in vitro NK-DC interactions have been explored. [21][22][23][24][25] In vivo depletion of NK cells resulted in marked reduction of tumor-specific CTL activity and suggests that the initiation of innate immune responses by NK cells may facilitate CTL development. 14 Interleukin-12 (IL-12) is a heterodimeric cytokine produced mainly by macrophages, monocytes, and DCs. ...
... B16-F10 cells were cultured with 250 U/ml mIFN-␥ for 24 hr before use as stimulator or target cells. Melanoma antigen associated peptides TRP-2 181-188 (VYDFFVWL) 66 and mGP100 [25][26][27][28][29][30][31][32][33] (EGSRN-QDWL) 67 were synthesized by ResGen (Huntsville, AL). Splenocytes were pulsed with TRP-2/mGP100 peptides (10 g/ml) at 37°C for 4 hr before 6-day cultivation. ...
In previous reports, systemic administration of a stimulatory monoclonal antibody directed against the 4-1BB receptor had no effect on survival or tumor burden in mice inoculated with the poorly immunogenic B16-F10 melanoma. We combined IL-12 gene transfer with 4-1BB costimulation to explore a previously noted cooperative anti-tumor effect against this model tumor. We hypothesize that the innate immune response mediated by IL-12-activated natural killer (NK) cells initiates the activation of the immune system, leading to the priming of T cells, whereas 4-1BB costimulation enhances the function of primed tumor-specific T cells. The effect of the combination therapy on the growth of subcutaneous (s.c.) tumors and pulmonary metastasis was examined. The combination therapy significantly retarded the growth of subcutaneously-inoculated tumors, and 50% of tumor-bearing mice survived with complete tumor regression. In contrast, neither IL-12 gene transfer nor anti-4-1BB antibody administration alone was as effective. Enhanced CTL activity against both B16-F10 tumor cells and TRP-2-pulsed EL4 syngeneic tumor cells was observed in tumor-bearing animals treated with the combination therapy 2 weeks after treatment and, in long-term survivors from this combination therapy, at >120 days. In a pulmonary metastatic model, only the combination therapy generated significant protection against metastasis. In vivo depletion of NK or CD8(+) but not CD4(+) subsets eliminated the protective immunity. Furthermore, NK cell depletion significantly reduced both tumor-specific CTL activity and the number of tumor-specific IFN-gamma-producing cells, suggesting that this synergistic effect requires the participation of both NK and CD8(+) T cells.
... Interactions between cell surface CD40 on antigen presenting cells and its ligand CD154 on NK cells and T cells also confers bidirectional activation [12]. This cell-contact mechanism is important in interactions between dendritic cells and NK cells [13,14], and increases NK cell toxicity [15], and the production of IL-12 during intracellular bacterial infection [16]. ...
... CD40/CD154 interactions have previously been identified as being important in macrophage activation, after interactions with T cells [14,17], as well as B cell maturation and immunoglobulin class switching, after interactions with NK cells [14]. Recent evidence has strengthened the role of CD40/CD154 interactions between NK cells and dendritic cells in producing dendritic cell maturation and activation, and this provides a mechanism for a genuine link between the innate and adaptive immune response following sepsis [12,13,20,21]. CD40/CD154 interaction between macrophages and NK cells was therefore a prime candidate as the mechanism of reciprocal activation of these cells during bacterial peritonitis. ...
Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.
... It has been reported that NK cells resemble T cells in their expression pattern of several co-stimulatory molecules such as CD28 or CD27. Blockage of CD28-CD80/86 or CD27-CD70 interaction between NK cells and DCs has been shown to reduce NK cell activation and cytotoxicity 60,61 . Interestingly, we found both costimulatory receptors CD80 and CD70 upregulated upon overnight incubation of TVH infected DCs. ...
The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8⁺ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.
... CD40L is upregulated on the surface of human and mouse NK cells when activated with IL-2 [83]. NK cells can be directly activated in an autocrine manner by their CD40L engagement with CD40 and this results in NK cell proliferation, activation and improved target killing [84][85][86]. ...
CD40 is a TNF receptor superfamily member expressed on both immune and non-immune cells. Interactions between B cell-expressed CD40 and its binding partner, CD40L, predominantly expressed on activated CD4+ T cells, play a critical role in promoting germinal center formation and the production of class-switched antibodies. Non-hematopoietic cells expressing CD40 can also engage CD40L and trigger a pro-inflammatory response. This article will highlight what is known about the biology of the CD40-CD40L axis in humans and describe the potential contribution of CD40 signaling on both hematopoietic and non-hematopoietic cells to autoimmune disease pathogenesis. Additionally, novel therapeutic approaches to target this pathway, currently being evaluated in clinical trials, are discussed.
... Both signaling mechanisms are mediated by cell-to-cell contact via simultaneous engagement of DCs transmembrane TNF and membrane-bound IL-15, with their respective NK cell receptors, leading to its activation. Furthermore, NK cells proliferation is also dependent on the interaction between CD40 and B7 molecules on DCs with CD40L and CD28 on NK cells, respectively [64]. IL-18 expressed by immature and mature DCs has also been implicated in NK cells activation. ...
Dendritic cells (DCs) and natural killer (NK) cells are both part of the innate immune system, also playing crucial functions in the regulation of adaptive immune responses. In recent years, numerous works have demonstrated that DCs and NK cells mutually influence each other with major consequences in the type and effectiveness of elicited immune responses. Among other effects, DC-NK crosstalk can result in NK cell activation and DC maturation or deletion, depending on its activation status. In this chapter and after a brief overview of DCs and NK immunobiology, we focus on the process of DC-NK crosstalk, highlighting the relevance of rationally exploring this interplay in the development of more effective cancer immunotherapy.
... The role of reciprocal activation between monocytes and NK cells in immune responses is gaining increased recognition (22)(23)(24)(25). NK cells function as one of the early sources of immunoregulatory cytokines (1). ...
Natural Killer (NK) cells are large granular lymphocytes that promote the antitumor response via communication with other cell types in the tumor microenvironment. Previously, we have shown that NK cells secrete a profile of immune stimulatory factors (e.g., IFNgamma, MIP-1alpha, TNFalpha) in response to dual stimulation with the combination of antibody (Ab)-coated tumor cells and cytokines, such as interleukin (IL) 12. We now demonstrate that this response is enhanced in the presence of autologous monocytes. Monocyte enhancement of NK cell activity was dependent on cell-to-cell contact as determined by a transwell assay. It was hypothesized that NK cell effector functions against Ab-coated tumor cells were enhanced via binding of MICA on monocytes to NK cell NKG2D receptors. Strategies to block MICA-NKG2D interactions resulted in reductions in IFNgamma production. Depletion of monocytes in vivo resulted in decreased IFNgamma production by murine NK cells upon exposure to Ab-coated tumor cells. In mice receiving trastuzumab and IL12 therapy, monocyte depletion resulted in significantly greater tumor growth in comparison to mock‑depleted controls (P < 0.05). These data suggest that NK cell-monocyte interactions enhance NK cell antitumor activity in the setting of monoclonal Ab therapy for cancer.
... DCs were shown to license NK cells by presenting IL-15 (14,22,35). To measure whether IL-32a would synergize or inhibit this property of IL-15, we stimulated NK cells with different concentrations of one or both cytokines and measured the production of the effector molecules granzyme B and perforin. ...
Cytokines produced by dendritic cells (DCs) can largely determine the direction of immunity. Transcriptional analysis revealed that besides IL-15, IL-32 was the only other cytokine expressed by human Langerhans cells. IL-32 is a human cytokine that exists in four main isoforms. Currently, little is known about the regulation and function of the various IL-32 isoforms. In this study, we found that IL-15 is a potent inducer of IL-32α in DCs. Because IL-15 promotes NK cell activation, we investigated the interplay between IL-32 and IL-15 and their role in NK cell activity. We show that IL-32α acts on NK cells to inhibit IL-15-mediated STAT5 phosphorylation and to suppress their IL-15-induced effector molecule expression and cytolytic capacity. IL-32α also acted on DCs by downregulating IL-15-induced IL-18 production, an important cytokine in NK cell activity. Blocking IL-32α during DC:NK cell coculture enhanced NK cell effector molecule expression as well as their cytolytic capacity. Taken together, our findings suggest a feedback inhibition of IL-15-mediated NK cell activity by IL-32α.
... Besides cytokines (IL-12, TNF, and IFNγ), mDCs can promote IFNγ production, CD69 expression, and cytotoxic functions of NK cells in a cell-to-cell contact-dependent manner. Ligands for the activating receptor NKG2D on NK cells, MICA and B, are expressed on human monocyte-derived DCs and contribute to CD69 expression on NK cells (58,62,63). ...
Regulation of T cell responses by innate lymphoid cells (ILCs) is increasingly documented and studied. Direct or indirect crosstalk between ILCs and T cells early during and after T cell activation can affect their differentiation, polarization, and survival. Natural killer (NK) cells that belong to the ILC1 group were initially described for their function in recognizing and eliminating “altered self” and as source of early inflammatory cytokines, most notably type II interferon. Using signals conveyed by various germ-line encoded activating and inhibitory receptors, NK cells are geared to sense sudden cellular changes that can be caused by infection events, malignant transformation, or cellular stress responses. T cells, when activated by TCR engagement (signal 1), costimulation (signal 2), and cytokines (signal 3), commit to a number of cellular alterations, including entry into rapid cell cycling, metabolic changes, and acquisition of effector functions. These abrupt changes may alert NK cells, and T cells might thereby expose themselves as NK cell targets. Here, we review how activated T cells can be recognized and regulated by NK cells and what consequences such regulation bears for T cell immunity in the context of vaccination, infection, or autoimmunity. Conversely, we will discuss mechanisms by which activated T cells protect themselves against NK cell attack and outline the significance of this safeguard mechanism.
... TLR9-dependent production of IL-12 is critical for PG545mediated NK cell activation in vivo. Among proinflammatory cytokines produced by DCs, IL-12 has been shown to be a critical mediator of NK cell activation (34)(35)(36)(37). We therefore examined whether PG545 caused DCs to produce IL-12 in vivo. ...
Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell-based antitumor therapies.
... It was recently reported by our group and others that murine DCs are capable of modulating innate immunity by stimulating NK cells through cell-to-cell contact (13,17). Thus we examined NK cell stimulation by tumor-pulsed DCs in two different experiments; determining IFN-production after co-culturing with purified splenic NK cells in vitro and NK cell activity after DC vaccination in vivo. ...
Immunization of dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. In this study, we examined whether the uptake of necrotic tumor cells could modulate DC phenotypes and whether the immunization of necrotic tumor cell-loaded DCs could elicit efficient tumor specific immune responses followed by a regression of established tumor burdens.
... Studien in vitro (Amakata et al., 2001; Gerosa et al., 2002 Gerosa et al., , 2005 Jinushi et al., 2003a) und in vivo (Fernandez et al., 1999; Krug et al., 2004; Orange and Biron, 1996) belegen sowohl im Rahmen der ...
Die vorliegende Arbeit beschäftigt sich mit der Frage, ob und welche bahnenden Faktoren für die Abstoßung von Klasse I MHC-defizienten Zellen benötigt werden.
Dabei wird erstmalig gezeigt, dass NK-Zellen für die Abstoßung von Klasse I MHC-defizienten "Selbst"-Zellen und Tumorzellen auf vorausgehende Bahnungsprozesse durch Dendritische Zellen angewiesen sind. Eine Schlüsselrolle in diesem engen Crosstalk zwischen NK-Zellen und Dendritischen Zellen nehmen dabei Typ-I-Interferone ein. Erst durch dieses komplexe Zusammenspiel können Natürliche Killerzellen ihre vollständige Effektorkompetenz gegenüber den Klasse I MHC-defizienten Zellen erlangen. Die vorliegenden Daten zeigen auch, dass die therapeutische Modulation der Aktivität Dendritischer Zellen ein interessantes Target für die NK-zell-vermittelte Immuntherapie von Tumoren darstellen könnte.
... Because NK cells can also indirectly contribute to tumor control by communicating with other immune cells (e.g., dendritic cells -DCs, NKT cells, macrophages, and T cells), there is an efficient adaptive antitumor response (12,21,49). Furthermore, it is well known from in vitro studies and colocalization experiments that NK cells can interact bidirectionally with DCs in areas of inflammation causing DC maturation, a consequent enhancement of NK cell function through positive feedback and exerting an influence on the polarization of primary T cell-responses toward a TH 1 response (50)(51)(52)(53)(54)(55). In fact, mature DCs can activate NK cell cytotoxicity and IFN-γ production, whereas activated NK cells are capable of enhancing DC maturation and IL-12 production. ...
Vaccine-based cancer immunotherapy has generated highly variable clinical results due to differing methods of vaccine preparation and variation in patient populations among other lesser factors. Moreover, these clinical responses do not necessarily correspond with the induction of tumor-specific cytotoxic lymphocytes. Here, we review the participation of natural killer (NK) cells as alternative immune components that could cooperate in successful vaccination treatment. NK cells have been described as helper cells in dendritic cell-based cancer vaccines, but the role in other kinds of vaccination strategies (whole cells, peptide, or DNA-based vaccines) is poorly understood. In this article, we address the following issues regarding the role of NK cells in cancer vaccines: NK cell anti-tumor action sites, and the loci of NK cell interaction with other immune cells; descriptions of new data on the memory characteristics of NK cells described in infectious diseases; and finally phenotypical and functional changes after vaccination measured by immunomonitoring in preclinical and clinical settings.
... Additionally, as we observed increased stimulation by BNBD3 when combined with LPS, secondary stimulation by BNBD3 may also be involved in the mechanism of activation/maturation of BMDCs (38). Another possibility for how the complexed vaccine might have affected the humoral responses, particularly with respect to the increase in levels of antibodies of the IgG2a isotype, could be from aBNBD3 directly acting on the natural killer (NK) cell subset (69) or indirectly acting on the iDCs with subsequent effects on the NK cell subset (70). Since activation of NK cells has been shown to increase antigen-specific IgG2a responses (71), the increased IgG2a levels that we observed might have occurred as a result of aBNBD3induced activity of NK cells. ...
Protective efficacy against bovine herpesvirus-1 (BoHV-1) has been demonstrated to be induced by a plasmid encoding the bovine beta-defensin, BNBD3, as a fusion with a truncated version of glycoprotein D (tgD). However, in spite of the increased cell-mediated immune responses induced by this DNA vaccine, the clinical responses of BoHV-1 challenged cattle were not reduced over those observed in animals vaccinated with the plasmid encoding tgD alone; this may have been because the vaccine failed to improve the humoral response. We hypothesized that an alternative vaccine design strategy, that utilized DNA vaccine pMASIA-tgD as a complex with BNBD3 peptide, might improve the humoral response while maintaining a robust Th1-type cell-mediated response. C57Bl/6 mice were vaccinated with pMASIA-tgD complexed with 0, 0.01875, 0.1875, or 1.875 nmol of a stable synthesized analog of BNBD3 (aBNBD3). The best results were seen in mice immunized with the vaccine comprised of pMASIA-tgD complexed to 0.1875 nmol aBNBD3. In this group the humoral response was improved as evidenced in increased VN, tgD-specific early IgG1 and later IgG2a; while the strong cell-mediated immune response, measured based on specific IFN-γ-secreting cells, was maintained relative to pMASIA-tgD. Modulation of the immune response may have been due in part to the effect of BNBD3 on dendritic cells (DCs). In vitro studies showed that murine bone marrow-derived DCs (BMDCs) pre-treated with aBNBD3 were activated as evidenced by down-regulated CD11c and were functionally matured as shown by increased allostimulatory ability. Native, synthesized and analog BNBD3 were equally capable of inducing functional maturation of BMDCs.
... Thus, our results may reflect a normal pathway that is important for the removal of Ab-coated targets.Interestingly, in our hands, monocytes enhanced NK cell secretion of IFN-γ in response to IL-12 alone(Figure 2.7a), suggesting a distinct need for this cellular compartment for the adequate production of cytokines by NK cells. Indeed, results of recent studies indicate that activation of NK cells following exposure to LPS or bacterial DNA is mediated by APC-derived IL-12, and that direct contact between APCs and NK cells is important for this response(107,108). Within the context oftumor immunity, Rodriguez-Calvillo et al. have shown that intratumoral IL-12 is largely responsible for the immunotherapeutic effects of NK cell activation in a CT-26 experimental tumor model (109). ...
... Thus, it is likely that other factors could be involved in the NK/DC cross-talk. Several cell surface receptors and soluble factors, such as IL-12, were shown to mediate the NK/DC cross-talk in humans and mice (21,46,(51)(52)(53)(54)(55). These factors, in addition to CD28/CTLA-4, might also be involved in regulating NK cell IFN-g production in response to mDCs in our experimental system. ...
NK cells express an array of activating and inhibitory receptors that determine NK cell responses upon triggering by cognate ligands. Although activating NK cell receptors recognize mainly ligands expressed by stressed, virus-infected, or transformed cells, most inhibitory receptors engage MHC class I, preventing NK cell activation in response to healthy cells. In this study, we provide insight into the regulation and function of additional receptors involved in mouse NK cell responses: CTLA-4 and CD28. CTLA-4 and CD28 engage the same ligands, B7-1 and B7-2, which are primarily expressed by APCs, such as dendritic cells. Our data demonstrate that activation of mouse NK cells with IL-2 induces the expression of CTLA-4 and upregulates CD28. CTLA-4 expression in IL-2-expanded NK cells was further up- or downregulated by IL-12 or TGF-β, respectively. Using gene-deficient NK cells, we show that CD28 induces, and CTLA-4 inhibits, IFN-γ release by NK cells upon engagement by the recombinant ligand, B7-1, or upon coculture with mature dendritic cells. Notably, we show that mouse NK cells infiltrating solid tumors express CD28 and CTLA-4 and respond to stimulation with recombinant B7-1, suggesting that the NK cell responses mediated by the CD28/CTLA-4:B7-1/B7-2 system could be of importance during malignant disease. Accordingly, our study might have implications for immunotherapy of cancer based on blocking anti-CTLA-4 mAbs.
... Recent data confirm that NK cells are required for the induction of potent anti-tumor-specific cytotoxic T lymphocytes (T cells) responses, by a mechanism involving dendritic cell (DC) editing (7,8). Furthermore, NK cells can recognize tumors that might evade T cell-mediated killing by aberrant human leukocyte antigen (HLA) expression (9), indicating that NK cells participate in tumor immunosurveillance. ...
Considerable evidence has been gathered over the last 10 years showing that the tumor microenvironment (TME) is not simply a passive recipient of immune cells, but an active participant in the establishment of immunosuppressive conditions. It is now well documented that hypoxia, within the TME, affects the functions of immune effectors including natural killer (NK) cells by multiple overlapping mechanisms. Indeed, each cell in the TME, irrespective of its transformation status, has the capacity to adapt to the hostile TME and produce immune modulatory signals or mediators affecting the function of immune cells either directly or through the stimulation of other cells present in the tumor site. This observation has led to intense research efforts focused mainly on tumor-derived factors. Notably, it has become increasingly clear that tumor cells secrete a number of environmental factors such as cytokines, growth factors, exosomes, and microRNAs impacting the immune cell response. Moreover, tumor cells in hostile microenvironments may activate their own intrinsic resistance mechanisms, such as autophagy, to escape the effective immune response. Such adaptive mechanisms may also include the ability of tumor cells to modify their metabolism and release several metabolites to impair the function of immune cells. In this review, we summarize the different mechanisms involved in the TME that affect the anti-tumor immune function of NK cells.
... In this model, NK cells lysed CD40 + adenocarcinoma cells in a contact dependent manner and this lysis was abrogated by blockade of the CD40-CD40L interaction. While few tumor immunotherapy models use CD40L agonists to activate NK cells, triggering of CD40L on the surface of NK cells by CD40 on APCs results in NK-cell proliferation [37,38]. Therefore, approaches that activate and expand NK cells in vivo through CD40L may be worth pursuing as potential tumor immunotherapy regimens. ...
CD40 ligation has been shown to induce antitumor effects in mice and cancer patients. Most of the studies have focused on the ability of an agonistic anti-CD40 mAb to either directly kill CD40-positive tumor cells or activate T-cell immune responses. In this review the authors focus on the ability of CD40 ligation to activate antitumor effector mechanisms of the cells of innate immunity such as macrophages and NK cells.
... Nonetheless, these levels remained significantly lower in comparison to healthy controls, indicating that ART therapy does not restore the functional competency of pDC and mdDC to induce IFN-c production by allogeneic NK cells. We and others [18,33,34] have demonstrated that pDC mediated activation of NK cells is partly mediated through secretion of IFN-a, whereas mdDC stimulate NK cells through an IL-12 dependent mechanism [35]. Therefore, we measured the levels of IFN-a and IL-12p70 in cell culture supernatants containing mature pDC and mdDC respectively. ...
Dendritic cells (DC) are potent inducers of natural killer (NK) cells. There are two distinct populations in blood, myeloid (mDC) and plasmacytoid (pDC) but they can also be generated In vitro from monocytes (mdDC). Although it is established that blood DC are lost in HIV-1 infection, the full impact of HIV-1 infection on DC-NK cell interactions remains elusive. We thus investigated the ability of pDC, mDC, and mdDC from viremic and anti-retroviral therapy-treated aviremic HIV-1+ patients to stimulate various NK cell functions. Stimulated pDC and mdDC from HIV-1+ patients showed reduced secretion of IFN-α and IL-12p70 respectively and their capacity to stimulate expression of CD25 and CD69, and IFN-γ secretion in NK cells was also reduced. pDC activation of NK cell degranulation in response to a tumour cell line was severely reduced in HIV-1+ patients but the ability of mDC to activate NK cells was not affected by HIV-1 infection, with the exception of HLA-DR induction. No differences were observed between viremic and aviremic patients indicating that anti-retroviral therapy had minimal effect on restoration on pDC and mdDC-mediated activation of NK cells. Results from this study provide further insight into HIV-1 mediated suppression of innate immune functions.
... Interferons act alone or can synergize with TNFa to inhibit proliferation of endothelial cells and angiogenesis12131415, and in this way act to prevent tumor development. Type I interferons also influence the immune system: they enhance the activity of natural killer cells that possess anti-tumor activity161718, and they promote the upregulation of class I MHC complexes and alter peptide presentation in nonimmune cells to emphasize their detection by the adaptive immune system [19,20]. Interferons thus impede tumorigenesis by several distinct mechanisms. ...
Local synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably.
To follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5.
Murine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth.
Our modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.
... NK cells are also a good source of GM-CSF releasing fairly large amounts upon activation either by cytokines or by engaging certain pathogens directly via TLRs (Blanchard et al., 1991; Boysen et al., 2006). This is important to further interactions with iDC since NK cells express CD40 ligand (CD154) and it is well known that GM-CSF participates in the up-regulation of CD40 on iDC (McLellan et al., 1996; Carbone et al., 1997; Amakata et al., 2001). These events set the stage for iDC:NK cell contact and cross-talk allowing for the full complement of innate mechanisms contributing to protection and leading to eventual acquired immunity. ...
Over the past several years, innate immunity has been recognized as having an important role as a front-line defense mechanism and as an integral part of the adaptive immune response. Innate immunity in cattle exposed to hemoparasites is spleen-dependent and age-related. In this review, we discuss general aspects of innate immunity and the cells involved in this aspect of the response to infection. We also provide examples of specific splenic regulatory and effector mechanisms involved in the response to Babesia bovis, an important tick-borne hemoparasitic disease of cattle. Evidence for the regulatory and effector role of bovine splenic monocytes and DC both in directing a type-1 response through interaction with splenic NK cells and γδT-cells will be presented.
... Our findings are consistent with the known capacity of IL-12 to activate NK cells and to stimulate them to secrete IFN-␥ (22). The critical role of IL-12 in DC-NK interaction is also supported in a recent report of human peripheral blood mononuclear cell-derived DC activation of NK cells (33). ...
Dendritic cells (DCs) are potent antigen-presenting cells with the potential for cancer immunotherapy. Adenoviral-mediated gene transfer is an attractive means to manipulate the immunostimulatory properties of DCs for therapeutic advantage. Because adenovirus induces DC maturation, we postulated that it would significantly alter their immune functions. Infected DCs markedly increased allogeneic and antigen-specific T-cell proliferation, and augmented natural killer cell lytic activity and IFN-gamma production. The enhanced effector cell stimulation by infected DCs was dependent on their secretion of interleukin 12. Immunization with infected DCs pulsed with tumor antigen protected against flank tumors in 78% of mice and induced a memory response. Antitumor immunity was dependent on both T cells and natural killer cells. Antigen-pulsed, mock-infected DCs were nonprotective. The findings that adenoviral vectors alone critically alter DC immune functions and antitumor properties have important implications for the design and interpretation of immunotherapy regimens using vector-based gene transfer to modulate immunity.
... The precise events or mediators that direct DC toward immunity or tolerance are not understood. Some have postulated that CD8␣ Ϫ (myeloid) DC are inherently immunogenic while CD8␣ ϩ (lymphoid) DC are tolerogenic (2)(3)(4). However, recent studies have indicated that CD8␣ ϩ and CD8␣ Ϫ DC are not distinct populations but simply represent different maturational stages of the same DC lineage (5). ...
Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c(+) and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-gamma, and TNF-alpha, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4(+) T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration.
... However, several recent reports confirm that DCs can activate NK cells and play an important role in crosstalk between innate and acquired immune responses. 45,46 We, therefore, attempted to analyze the mechanism behind the anti-B16BL6 tumor effects of immunization with AdRGD-gp100/mBM-DCs. Splenocytes from mice immunized with AdRGD-gp100/mBM-DCs could injure Antimelanoma effect of DCs transduced by AdRGD-gp100 N Okada et al YAC-1 cells more effectively than those from intact mice, whereas NK activity did not change upon immunization with Ad-gp100/mBM-DCs or intact mBM-DCs. Although data were not shown, we confirmed that the increase in NK activity in mice immunized with AdRGD-gp100/mBM-DCs disappeared 2 weeks postimmunization. ...
Dendritic cells (DCs) are the most potent professional antigen-presenting cells for the initiation of antigen-specific immune responses, and antigen-loaded DCs have been regarded as promising vaccines in cancer immunotherapy. We previously demonstrated that RGD fiber-mutant adenovirus vector (AdRGD) could attain highly efficient gene transduction into human and murine DCs. The aim of the present study is to demonstrate the predominance of ex vivo genetic DC manipulation using AdRGD in improving the efficacy of DC-based immunotherapy targeting gp100, a melanoma-associated antigen (MAA). Vaccination with murine bone marrow-derived DCs transduced with AdRGD encoding gp100 (AdRGD-gp100/mBM-DCs) dramatically improved resistance to B16BL6 melanoma challenge and pulmonary metastasis as compared with immunization with conventional Ad-gp100-transduced mBM-DCs. The improvement in antimelanoma effects upon immunization with AdRGD-gp100/mBM-DCs correlated with enhanced cytotoxic activities of natural killer (NK) cells and B16BL6-specific cytotoxic T lymphocytes (CTLs). Furthermore, in vivo depletion analysis demonstrated that CD8(+) CTLs and NK cells were the predominant effector cells responsible for the anti-B16BL6 immunity induced by vaccination with AdRGD-gp100/mBM-DCs, and that helper function of CD4(+) T cells was necessary for sufficiently eliciting effector activity. These findings clearly revealed that highly efficient MAA gene transduction to DCs by AdRGD could greatly improve the efficacy of DC-based immunotherapy against melanoma.
Peptide vaccines have shown great potential in cancer immunotherapy by targeting tumor antigens and activating the patient's immune system to mount a specific response against cancer cells. However, the efficacy of peptide vaccines in inducing a sustained immune response and achieving clinical benefit remains a major challenge. In this review, we discuss the current status of peptide vaccines in cancer immunotherapy and strategies to improve their efficacy. We summarize the recent advancements in the development of peptide vaccines in pre-clinical and clinical settings, including the use of novel adjuvants, neoantigens, nano-delivery systems, and combination therapies. We also highlight the importance of personalized cancer vaccines, which consider the unique genetic and immunological profiles of individual patients. We also discuss the strategies to enhance the immunogenicity of peptide vaccines such as multivalent peptides, conjugated peptides, fusion proteins, and self-assembled peptides. Although, peptide vaccines alone are weak immunogens, combining peptide vaccines with other immunotherapeutic approaches and developing novel approaches such as personalized vaccines can be promising methods to significantly enhance their efficacy and improve the clinical outcomes for cancer patients.
The role of autophagy in cancer is complex and is likely dependent on tumor type, stage, and genetic context. This complexity is illustrated by the identification of settings where autophagy acts potently to either promote or inhibit tumorigenesis. It is now well established that autophagy serves as a barrier to limit tumor initiation; however, once tumors are well established, autophagy plays a positive role in malignant progression and in subsequent tumor maintenance. Autophagy is also induced in response to cancer therapies where it can function as a survival mechanism and limit drug efficacy. Such findings have inspired significant interest in applying anti-autophagy therapies as an entirely new approach to cancer treatment. While much remains to be learned about the regulation and context-dependent biological role of autophagy, it is now widely established that modulation of this process will be an attractive avenue for future anticancer therapeutic approaches.
In this chapter, we will summarize recent data describing how autophagy activation under hypoxic stress impairs the antitumor immune response and compromises cancer immunotherapeutic approaches. In addition, we will discuss how tumor manages to hide from the immune attack, and either mounts a “counterattack” or develops resistance to immune cells. In particular, we will focus on the effect of hypoxia-induced autophagy in allowing tumor cells to outmaneuver an effective immune response and escape from immune surveillance. It is our belief that autophagy may represent a conceptual realm for new immunotherapeutic strategies aiming to block immune escape and therefore providing rational approach to future tumor immunotherapy design.
Natural killer (NK) cells represent a distinct lineage of lymphocyte that is conserved in all mammals. Their dominant role is to protect against infection prior to the appearance of effective T cell responses. They may also be important, however, to antitumor responses, and to coordination of adaptive immunity. Functionally, they are much like cytotoxic T cells in using granules containing perforin and granzymes to kill target cells; they also secrete many immune-coordinating cytokines, foremost of which is interferon-?. Recent studies show NK cells to be more complex than previously suspected - transiting a multisite and multistage developmental process, engaging in critical interactions with accessory cells, and using a panoply of receptors to recognize targets. This chapter summarizes these elements of NK biology, in addition to NK cell assays and chemical agents that modulate NK function.
Inflammatory bowel disease (IBD) is closely associated with the abnormal immune response of intestinal immune system to enterobacteria and dietary antigens. Large amounts of mononuclear lymphocyte infiltrates and high expression levels of proinflammatory cytokines and costimulatory molecules are present in inflamed mucosa. NK cells, involved in innate and acquired immune response, have cytolitic activities through secretion of lytic proteins, and produce proinflammatory mediators. Increased infiltration of NK cells present in inflamed mucosa of IBD expresses high levels of activated molecules and proinflammatory cytokines, and plays a role in intestinal mucosal damage.
Endocannabinoïds, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), belong to the andocannabinoïd system are involved in regulating of many biological systems. They have a role in modulating the immune system and anti-tumor defense. In this study, we show that AEA and 2-AG may modulate the activation of natural killer cells CD56+CD3- (NK) cells and NK92. At low doses , they potentiate the production of IFN gamma induced by suboptimal doses of IL-12, IL-15 and IL-2. In addition, AEA and 2-AG potentiate the cytotoxicity of NK cells. Experiments to quantify transcripts encoding FasL, TRAIL, perforin and granzyme and inhibition experiments indicate that this increase in cytotoxicity was correlated with increased expression of FasL, while production of granzyme, perforin and expression TRAIL remains unchanged. Endocannabinoïd effects on NK cells are inhibited in the presence of antagonist of the type 2 endocannabinoïd receptor (CB2). These in vitro studies have been confirmed in vivo in a murine tumor model. Murine NK cells stimulated ex-vivo by endocannabinoïds and reinjected directly in the tumor, induce a significant slowing of tumor growth. These results show for the first time that endocannabinoïds AEA and 2-AG used at low doses are involved in the modulation of regulatory activities (objectified by the production of IFN gamma) and cytotoxic activities of NK cells.
In this brief review the advances and hurdles of the modern-day ACT (adoptive cell transfer) immunotherapy of cancer are discussed, with the focus on the positive or negative role of CD4+ T helper lymphocytes as one of major constituents of oncologic patient-administered CIK (cytokine- induced killers) lymphocyte culture. The beneficial role of CD4+ T helpers in adoptively-transferred lymphocyte culture is considered, questioned and being put under doubt. “Infectious tolerance” and tumor “immune avoidance” phenomena are described, emphasizing on their dramatic implications for cancer ACT therapy. The ways to circumvent apparent undesired effects of CD4+ T helpers elevated presence in CIK bulk mass are discussed, such as complete removal of CD4 – positive cells, along with a less radical measure, which is depletion of CD4+CD25+FoxP3+ T regulatory lymphocytes from bulk CIK culture.
In this brief review the advances and hurdles of the modern-day ACT (adoptive cell transfer) immunotherapy of cancer are discussed, with the focus on the positive or negative role of CD4+ T helper lymphocytes as one of major constituents of oncologic patient-administered CIK (cytokine-induced killers) lymphocyte culture. The beneficial role of CD4+ T helpers in adoptively-transferred lymphocyte culture is considered, questioned and being put under doubt. “Infectious tolerance” and tumor “immune avoidance” phenomena are described, emphasizing on their dramatic implications for cancer ACT therapy. The ways to circumvent apparent undesired effects of CD4+ T helpers elevated presence in CIK bulk mass are discussed, such as complete removal of CD4 -positive cells, along with a less radical measure, which is depletion of CD4+CD25+FoxP3+ T regulatory lymphocytes from bulk CIK culture.
Human blood natural killer (NK) cells exert strong cytotoxicity against transformed cells and produce different cytokines and chemokines with an important role in modulating immune responses. However, the nature of NK cell function depends on NK cell interaction with other immune cells. One type of immune cells that communicate with NK cells are 6-sulfo LacNAc dendritic cells (slanDCs), which comprise a major subpopulation of proinflammatory human blood myeloid DCs. In this study, we investigated the molecular mechanisms by which slanDCs interact with NK cells. Our in vitro studies demonstrate that lipopolysaccharide (LPS) stimulated slanDCs enhance activation and function of NK cells essentially via membrane-bound TNF-α (mTNF-α). LPS stimulation upregulates expression of mTNF-α in slanDCs, and surface tumor necrosis factor receptor 2 (TNFR2) is upregulated on NK cells after coincubation with slanDCs. Interleukin (IL)-12 secreted by slanDCs increases surface expression of TNFR2 in NK cells. TNFR2 signaling in NK cells leads to activation of NF-kB, a transcription factor for cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF provided by NK cells is responsible for enhancing IL-12 secretion in slanDCs. In conclusion, TNFR2- and IL-12-signaling, which support one another, enables slanDCs to enhance NK cell function through mTNF-α, thereby regulating immune responses.This article is protected by copyright. All rights reserved
Migration and localization of natural killer (NK) cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-dendritic cell (DC) crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and lipopolysaccharide (LPS)-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony stimulating factor (GM-CSF) in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumour and infections. This article is protected by copyright. All rights reserved.
A successful pregnancy relies on immunological adaptations that allow the fetus to grow and develop in the uterus, despite being recognized by maternal immune cells. Among several immunocompetent cell types present within the human maternal/fetal interface, DC-SIGN(+) dendritic cells (DCs) and CD56(+) natural killer (NK) cells are of major importance for early pregnancy maintenance, not only generating maternal immunological tolerance but also regulating stromal cell differentiation. Previous reports show the presence of NK-DC cell conjugates in first trimester human decidua, suggesting that these cells may play a role in the modulation of the local immune response within the uterus. While effective immunity is necessary to protect the mother from harmful pathogens, some form of tolerance must be activated to avoid an immune response against fetal antigens. This review article discusses current evidence concerning the functions of DC and NK cells in pregnancy and their liaison in human decidua.Cellular & Molecular Immunology advance online publication, 23 June 2014; doi:10.1038/cmi.2014.36.
Galectin-9 (Gal-9) inhibits the metastasis of tumor cells by blocking their adhesion to endothelium and the extracellular matrix. In this study, we addressed the involvement of Gal-9 in anti-tumor activity. Gal-9 significantly prolonged the survival of B16F10 melanoma-bearing mice. Gal-9 increased the numbers of NK cells, CD8 T cells and macrophages in tumor-bearing mice. Gal-9-mediated anti-tumor activity was not induced in NK cell-, macrophage- and CD8 T cell-depleted mice. NK cells from Gal-9-treated mice, compared to PBS-treated mice, exhibited significantly higher cytolytic activity. Co-culture of naïve NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity. We also found that Ly-6C+CD11b+F4/80+ macrophages with plasmacytoid cell (pDC)-like phenotypes (PDCA-1 and B220) were responsible for the enhanced NK activity. These results provide evidence that Gal-9 promotes NK cell-mediated anti-tumor activity by expanding unique macrophages with a pDC-like phenotype.
Dendritic cells (DCs) are professional antigen-presenting cells, which display an extraordinary capacity to induce T-cell responses. Recent findings revealed that DCs also play a crucial role in the activation of natural killer (NK) cells representing important effectors in the innate immune defense against viruses and tumors. Here, we summarize various studies investigating the bidirectional crosstalk between human DCs and NK cells. In this context, it has been reported that DCs efficiently enhance CD69 expression, proliferation, interferon (IFN)-γ secretion and cytotoxic activity of NK cells. Cell membrane-associated molecules as well as soluble factors such as interleukin-12, tumor necrosis factor-α and type I IFNs contributed to DC-mediated NK cell activation. Reciprocally, the ability of human NK cells to enhance the immunostimulatory capacity of DCs was shown. Thus, NK cells promoted the maturation of DCs and markedly augmented their capacity to produce proinflammatory cytokines and to stimulate T-cell responses. The NK cell-mediated effects on DCs were dependent on cell membrane-associated molecules such as NKp30 and soluble factors such as tumor necrosis factor-α and IFN-γ. In conclusion, the reciprocal activating interaction between human DCs and NK cells may play a pivotal role in the immune defense against viruses and tumors.
Dendritic cells (DC) were originally found critical in the setting of cognate immune responses. We first demonstrated that DC can also induce mouse NK cell activation and NK cell dependent-antitumor effects in mice. Here we analyzed the dynamics between DC and NK cells in human in vitro model systems. In the absence of LPS, DC do not trigger resting NK cells. Conversely, in the presence of LPS, resting bulk NK cells interacting with DC acquire CD25 and CD69 surface expression, produce high levels of IFN-gamma and lyse DAUDI cells. On activated IL-2 dependent NK cell lines, regardless of their differentiation stage, DC maintain or enhance NK cell proliferation and effector functions in the absence of exogenous cytokines. While IL-12, IL-15 and IL-18 are not critical, a direct cell-to-cell contact is mandatory for NK activation by DC and required for optimal proliferation. These data imply that DC also modulate human NK cell innate effector functions.
The anti-tumor activity of recombinant mAb's directed against tumor cell growth receptors has generally been considered to result from direct antiproliferative effects, the induction of apoptosis, or possibly Ab-dependent cellular cytotoxicity mediated against tumor targets. However, it remains unclear to what degree these mechanisms actually aid in the clearance of Ab-coated tumor cells in vivo. We show here that NK cells secrete a distinct profile of potent immunostimulatory cytokines in response to dual stimulation with Ab-coated tumor cells and IL-12. This response could not be duplicated by costimulation with other ILs and was significantly enhanced in the presence of monocytes. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (FcgammaRIII) and the IL-12 receptor expressed on NK cells. Coadministration of Ab-coated tumor cells and IL-12 to BALB/c mice resulted in enhanced circulating levels of NK cell-derived cytokines with the capacity to augment anti-tumor immunity. These findings suggest that, in addition to mediating cellular cytotoxicity and apoptosis, the anti-tumor activity of mAb's might also result from activation of a potent cytokine secretion program within immune effectors capable of recognizing mAb-coated targets.
There are a surprisingly large number of human natural killer (NK) cell deficiency states that provide insight into the role of NK cells in defense against human infectious disease. Many disorders associated with NK cell defects are caused by single gene mutations and, thus, give additional understanding concerning the function of specific molecules in NK cell development and activities. A resounding theme of NK cell deficiencies is susceptibility to herpesviruses, suggesting that unexplained severe herpesviral infection should raise the possibility of an NK cell deficit.
Dendritic cells (DCs) augment effector functions of NK cells, but the underlying mechanisms are not fully understood. Here we show in an in vitro coculture system that human monocyte-derived DCs enhance IFN-gamma production, CD69 expression, and K562 cytolytic ability of NK cells when DCs are prestimulated with various maturation stimuli such as IFN-alpha or LPS. Of interest is the finding that NK cell activation mediated by LPS-stimulated DCs was dependent on IL-12 produced in DC/NK coculture, but that IFN-alpha-stimulated DC-mediated activation was not. Alternatively, MHC class I-related chain A and B (MICA/B), ligands for NKG2D activating receptor, were found to be induced on DCs upon IFN-alpha stimulation and to be responsible for the NK activation because mAb-mediated masking of MICA/B as well as inhibition of direct cell-to-cell contact using transwell insert completely abolished DC-dependent NK cell activation by IFN-alpha. Finally, DCs recovered from chronic hepatitis C virus-infected patients showed defects in the induction of MICA/B and impaired ability to activate NK cells in response to IFN-alpha stimulation. These findings suggested that MICA/B induction on DCs may be one of the mechanisms by which IFN-alpha activates NK cells; this impairment might affect IFN-alpha responsiveness in hepatitis C virus infection.
Natural killer (NK) cells have a well-established role in host defense against viral infections and malignancies. However, their function in bacterial infection and sepsis is poorly defined. We hypothesized that NK cells, as a major producer of interferon-gamma during sepsis, would be important in host defense against bacterial infections. Cecal ligation and puncture (CLP) was performed on Swiss Webster mice depleted of NK cells by pretreatment with anti-asialo GM1 and control mice given immunoglobulin G (IgG) antibody. NK cell-depleted mice had significantly higher anaerobic bacterial counts in the liver and peritoneal lavage fluid, as well as higher aerobic counts in the liver and blood 4 h after CLP. Macrophage phagocytosis, nitric oxide production, and interleukin (IL)-6 levels at 4 h were also decreased in mice depleted of NK cells compared with controls. Greater neutrophil influx into the peritoneum, indicated by higher myeloperoxidase levels, was also seen in NK cell-depleted mice. At 8 and 18 h after CLP, bacterial counts were similar between groups, and overall survival rates were not significantly different. Peritoneal IL-12 levels significantly increased by 18 h in normal mice, but not in NK cell-depleted animals. Our data suggest that NK cells participate in the early local and systemic eradication of bacteria and regulation of IL-12 during polymicrobial sepsis. These effects are likely due to their interactions with macrophages.
Dendritic cells (DC) are initiators of T cell-mediated immunity. However, less is known about the relationship between DC and natural killer (NK) cells, and direct evidence of their interaction in vivo is scarce. Interleukin (IL)-12 is an activator of both DC and NK cells. We postulated that secretion of IL-12 by DC would enable them to activate NK cells. Bone marrow-derived DC propagated only in granulocyte macrophage colony-stimulating factor did not activate NK cells. In contrast, DC engineered to express IL-12 markedly stimulated NK cells as determined by coculture experiments in vitro, assays of NK cells isolated from treated animals, and survival experiments in a systemic tumor model. Activation depended on both DC-NK cellular interaction and secretion of IL-12. Adoptive transfer of DC expressing IL-12 to mice markedly increased NK cell interferon-gamma production and lytic activity in vivo. Treated mice were also protected against B16 melanoma hepatic metastases. The in vivo effects on NK cells were DC-specific. Administration of IL-12 protein alone or melanoma cells or fibroblasts engineered to secrete IL-12 were only weakly activating. Our findings demonstrate that IL-12 expression by DC enables them to activate NK cells and provide evidence for a substantial DC-NK relationship in vivo.
Natural killer (NK) cells are major cytokine producers during bacterial sepsis, but their precise role is undefined. This study investigates the effect of NK cell depletion with and without prior activation on macrophage function and bacterial clearance during cecal ligation and puncture. Two different NK cell-depleting antibodies were used: anti-asialo-GM1 (GM1), a nonactivating antibody, and anti-NK1.1 (NK1.1), an NK cell-activating antibody. C57BL/6 mice were NK depleted with either GM1 or NK1.1 by intraperitoneal injection 7 and 3 days before experimentation. Control animals received isotype immunoglobulin G. Depletion was confirmed by flow cytometry. Bacterial levels in peritoneal washout, blood, and liver were determined 4 hours after cecal ligation and puncture. Macrophage activation was measured by phagocytosis ability and by production of nitric oxide and interleukin-6. Depletion with GM1 resulted in significantly higher bacterial levels at 4 hours, whereas depletion with NK1.1 had the opposite effect of significantly decreasing bacterial levels. Macrophage phagocytosis ability was significantly increased in mice depleted with NK1.1 compared with those mice depleted with GM1. We conclude that activation of NK cells improves bacterial clearance by priming macrophages to help clear a subsequent bacterial challenge. Macrophages are less able to clear bacteria when NK cells are depleted without activation. NK cells are therefore important in bacterial clearance through interactions with macrophages.
Dendritic cells (DC) were initially described as antigen presenting cells able to prime naive T cells. In fact, dendritic cells are also able to activate natural killer (NK) cells. The crosstalk between DC and NK is now regarded as a main stage in the initiation of innate and adaptative immune responses. Moreover, DC mediated-NK cell activation is particularly efficient since DC promote both effector functions and survival or proliferation of NK cells. Furthermore, recent publications in the field of NK cell biology underscore a role for NK cells in anti-tumor effects. NK cell-based immunotherapy should be reconsidered Here, we will report how pioneering immunotherapy trials based on natural cytotoxicity and progress in NK cell biology can promote NK cells in anti-tumor immune responses. Then, we will discuss the biological features of the DC/NK crosstalk and its implementations in immunotherapy protocols.
John Libbey Eurotext 2003
Some mouse strains produce strong pro-inflammatory, T-helper (Th)1 responses (e.g. C57BL/6), or strong anti-inflammatory, Th2 responses (e.g. BALB/c). The exact mechanisms for development of distinct immune responses to infection are not completely understood, although cytokines such as interleukin (IL)-12, IL-18 and IL-4 are known to play roles. Natural killer T (NKT)/natural killer (NK) cells are important regulators of immune responses in infection and non-infection models, and NKT/NK activation is also regulated by IL-12 and IL-18 in many models. We investigated the role of IL-12/IL-18 in NKT/NK activation in murine bacterial peritonitis, as well as differential NKT and NK cell activation in C57BL/6 and BALB/c mice. No differences in NKT or NK cell activation or intracellular interferon (IFN)-gamma were determined between mice given control, anti-IL-12 or anti-IL-18 antibodies or in NKT/NK cell activation in STAT4-/- mice (deficient in IL-12 signaling) or wild type controls. However, there were significant differences in the activation of NKT and NK cells between C57BL/6 mice and BALB/c mice, with NKT/NK cytokine production following Th1 or Th2 lines dependent on strain. This suggests a role for NKT and NK cell activation in the development of Th1 and Th2 responses during bacterial infection independently of IL-12 or IL-18.
The aim of this study was to analyse whether split influenza vaccine may elicit NK cytotoxic response in the vaccinated elderly people and whether this effect may be maintained over few weeks after vaccination. It was also worth investigating the relation between NK activity in the vaccinated and specific immune protection against influenza and non-specific against other infections. Two groups of volunteers were vaccinated with trivalent split viron influenza vaccine in two consecutive seasons (1999/2000; 2000/2001). The elderly group consisted of 142 people (65-92 years old) in the first season and 110 in the second; while the young (16-44 years old) of 98 and 67 people, respectively. An analysis of NK cytotoxic activity had been done before vaccination, two days, one month and fifth months thereafter. The results revealed that vaccination with the influenza vaccine had an augmenting effect on NK activity, in all groups examined, in both epidemic seasons, visible at two days and 1 month after the vaccination. In the elderly high pre- and post-vaccination NK activity was related to higher titers of anti-hemagglutinin, better health status and lower incidence of all cause respiratory tract infections. At the second vaccination, most of the elderly with chronic medical conditions and high NK activity, who did not attain the protective level of anti-hemagglutinins in the first season, converted into the protected. High pre- and post-vaccination NK activity predisposes elderly people to the protective humoral anti-hemagglutinin response and gives better protection from respiratory tract infections.
We investigated the possibility that T helper cells might enhance the stimulatory function of dendritic cells (DCs). We found that ligation of CD40 by CD40L triggers the production of extremely high levels ofbioactive IL-12. Other stimuli such as microbial agents, TNF-cx or LPS are much less effective or not at all. In addition, CD40L is the most potent stimulus in upregu-lating the expression ofICAM-1, CD80, and CD86 molecules on DCs. These effects of CD40 ligation result in an increased capacity of DCs to trigger proliferative responses and IFN-~/pro-duction by T cells. These findings reveal a new role for CD40-CD40L interaction in regulating DC function and are relevant to design therapeutic strategies using cultured DCs.
The sympathetic nervous system has been implicated in mediating stress-induced alterations in NK cell activity, particularly through stimulation of beta-adrenergic receptors. However, because catecholamines induce time-dependent alterations in the distribution of NK cells, the impact of beta-adrenergic stimulation on individual NK cell cytotoxicity is not clear, nor are its implications regarding host resistance to metastatic spread. To address these issues, we used the beta-adrenergic agonist, metaproterenol (MP), in F344 rats. The number of blood NK cells doubled within 10 min of MP administration and returned to baseline levels within 1 h. By this time, MP suppressed blood NK activity in a dose-dependent manner. Two beta-adrenergic antagonists, propranolol, which crosses the blood-brain barrier, and nadolol, which does not, blocked this suppression. Corresponding findings were obtained using an NK-sensitive tumor model, the MADB106. MP caused an up to 10 times increase in the number of tumor cells retained in the lungs 1 day after inoculation and a similar rise in the number of consequent lung metastases detected 3 wk later. These effects were dose dependent and nadolol reversible. NK cells appear to play a central role in mediating the tumor-enhancing effects of MP because their selective depletion nearly abolished this effect. Overall, our findings suggest that independent of the transitory increase in numbers of blood NK cells, in vivo beta-adrenergic stimulation suppresses NK activity in the rat. This suppression is induced peripherally and can compromise host resistance to NK-sensitive tumors. Homologies to studies in humans and clinical relevance are discussed.
Monocyte-specific monoclonal antibodies (7) were used to compare the efficacy of monocytes and dendritic cells as accessory or stimulator cells for human T cell replication. Both unfractionated and plastic-adherent mononuclear cells were first treated with a cytolytic antimonocyte antibody that kills greater than 95% of monocytes but not dendritic cells. When tested as stimulators of the mixed leukocyte reaction (MLR) and of oxidative mitogenesis (the proliferation of T cells modified with sodium periodate), the monocyte-depleted cells had normal or enhanced stimulatory capacity. Monocyte-depleted mononuclear cells also proliferated normally to soluble antigens (Candida albicans, tetanus toxoid), even under limiting conditions of cell dose, antigen dose, and culture time. Adherent blood mononuclear cells were next separated into monocyte-enriched and -depleted components using fluoresceinated antimonocyte antibody and the cell sorter. The depleted fraction (less than 2% monocytes by esterase staining and by cytology) contained the dendritic cells and exhibited at least 75% of the accessory activity. The monocyte-rich fraction (approximately 97% esterase positive) stimulated the MLR and oxidative mitogenesis weakly, and was comparable in potency to nonadherent cells. Cell-specific antibodies and complement were also used to prepare dendritic cells that were thoroughly depleted of monocytes and lymphocytes. The dendritic cells (70-80% pure) were potent stimulators of the allogeneic MLR, syngeneic MLR, and tetanus toxoid response, being active at stimulator to responder ratios of 1:100 or less. Taken together with previous studies (1, 2), these experiments indicate that the dendritic cell is the major stimulator of T cell replication in man. The contribution of class II products of the major histocompatibility complex (7) was then evaluated with a new monoclonal, 9.3F10. Accessory function was dramatically inhibited if cells bearing class II antigens were killed with 9.3F10 and complement, or if class II molecules were blocked by the addition of 9.3F10 Fab to the culture medium. The expression of 9.3F10 class II products was therefore studied on purified monocytes and dendritic cells. Most if not all cells in both populations reacted with 9.3F10, and each population exhibited approximately 150,000 125I-Fab 9.3F10 binding sites per cell. Since Ia+ dendritic cells are active accessory cells, but Ia+ monocytes are not, class II products are necessary but not sufficient for the stimulation of T cell proliferation in man.
Cytotoxic lymphocytes are typically generated from unfractionated suspensions of human lymphocytes by stimulating with heterogeneous APCs and exogeneous growth factors. We have found that human blood dendritic cells can directly stimulate allogeneic human CD8+ T cells to proliferate and express antigen-specific cytotoxic activity. These primary responses, which are accompanied by the release of T cell growth factor(s), are induced in the absence of CD4+ helper T cells and are not inhibited by anti-CD4 mAb. Both antigen-specific CTL as well as nonspecific NK cells can be elicited by dendritic cells. The NK cell response can be depleted at the precursor level by panning with an anti-CD11b mAb, which removes a CD11b+/CD28-, CD16+ subset from the starting CD4- responders. Allogeneic blood monocytes are neither stimulatory nor inhibitory of these primary CD4- MLRs, even though monocytes present alloantigen in such a way as to be recognized as specific targets for CTL that have been sensitized by dendritic cells. The number of CD8+ cells that are blast transformed and express an activated phenotype (i.e., HLA DR/DQ+, CD25/IL-2R+, CD45R-) reaches 30-40% of the culture at day 4-5, the peak of the helper-independent response. We conclude that antigen-presentation by dendritic cells is sufficient in itself to prime cytolytic precursors. We speculate that using dendritic cell stimulators and CD4- responders in MLRs may be more efficient than standard tissue typing approaches for the detection of subtle, but important class I MHC-restricted histoincompatibilities in human transplantation.
T cells recognize peptides that are bound to MHC molecules on the surface of different types of antigen-presenting cells (APC). Antigen presentation most often is studied using T cells that have undergone priming in situ, or cell lines that have been chronically stimulated in vitro. The use of primed cells provides sufficient numbers of antigen-reactive lymphocytes for experimental study. A more complete understanding of immunogenicity, however, requires that one develop systems for studying the onset of a T cell response from unprimed lymphocytes, especially in situ. Here it is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC. The dendritic cells were isolated from spleen, pulsed with protein antigens, and then administered to naive mice. Antigen-responsive T cells developed in the draining lymphoid tissue, and these T cells only recognized protein when presented on cells bearing the same MHC products as the original priming dendritic cells. In contrast, little or no priming was seen if antigen-pulsed spleen cells or peritoneal cells were injected. Since very small amounts of the foreign protein were visualized within endocytic vacuoles of antigen-pulsed dendritic cells, it is suggested that dendritic cells have a small but relevant vacuolar system for presenting antigens over a several day period in situ.
We have identified and purified a novel cytokine, NK cell stimulatory factor (NKSF), from the cell-free supernatant fluid of the phorbol diester-induced EBV-transformed human B lymphoblastoid cell line RPMI 8866. NKSF activity is mostly associated to a 70-kD anionic glycoprotein. The purified 70-kD protein, isolated from an SDS-PAGE gel, yields upon reduction two small species of molecular masses of 40 and 35 kD, suggesting that this cytokine is a heterodimer. When added to human PBL, purified NKSF preparations induce IFN-gamma production and synergize with rIL-2 in this activity, augment the NK cell-mediated cytotoxicity of PBL preparations against both NK-sensitive and NK-resistant target cell lines, and enhance the mitogenic response of T cells to mitogenic lectins and phorbol diesters. The three activities remain associated through different purification steps resulting in a 9,200-fold purification, and purified NKSF mediates the three biological activities at concentrations in the range of 0.1-10 pM. These data strongly suggest that the same molecule mediates these three activities, although the presence of traces of contaminant peptides even in the most purified NKSF preparations does not allow us to exclude the possibility that distinct biologically active molecules have been co-purified. The absence of other known cytokines in the purified NKSF preparations, the unusual molecular conformation of NKSF, the high specific activity of the purified protein, and the spectrum of biological activities distinguish NKSF from other previously described cytokines.
Highly enriched populations of B lymphoblasts have been isolated after culture with anti-Ig-Sepharose and compared with dendritic cells as stimulators of CD4+ T cells in the murine MLR. The two populations clearly differed in phenotype; anti-Ig blasts were FcR+, B220+, 33D1-, while dendritic cells were FcR-, B220-, 33D1+. However, as MLR stimulators, they shared many common features. Both cells (a) expressed comparable levels of class II MHC products; (b) independently stimulated the primary MLR and the production of several T derived lymphokines including IL-2 and IL-4; and (c) were comparable in stimulating freshly sensitized T cells. However, the relative potencies of dendritic cells and anti-Ig blasts as primary MLR stimulators varied in a strain-dependent fashion. Only anti-Ig blasts could stimulate across an Mls barrier, being at least 100 times more active in stimulating Mls-mismatched, MHC-matched T cells, relative to syngeneic T cells. In contrast, dendritic cells were 10-30 times more potent than anti-Ig blasts when stimulating across an MHC barrier and were likewise more effective in binding MHC-disparate T cells to form the clusters in which the MLR was generated. Dendritic cell-T cell clustering was resistant to anti-LFA-1 mAb, while B blast-T cell clustering was totally blocked. Thus, anti-Ig B lymphoblasts and dendritic cells, two cell types which differ markedly in phenotype, also differ in efficiency and mechanism for initiating responses in allogeneic T cells.
To study the role of the CD40-CD40 ligand interaction in the development of memory B cells and its level of action during primary antibody responses in vivo, mice were injected with a soluble CD40 fusion protein (sCD40-gamma 1), so as to block the interaction. The effects of the treatment on the primary antibody response were reminiscent of hyper-immunoglobulin M (IgM) syndrome (HIMG1): antigen-specific IgG responses were grossly inhibited whereas the IgM response was augmented severalfold. The latter observation suggests that there is a T-dependent, CD40 ligand-independent pathway of B cell activation that leads to IgM responses and that a significant component of the IgM in HIMG1 patients is derived from T-dependent responses. The secondary response was not readily blocked by sCD40-gamma 1 treatment, indicating a relative independence of CD40 ligation of antigen-experienced B cells. The most striking finding from these studies is that the development of memory B cell populations (measured by adoptive transfer) is grossly impaired by administration of sCD40-gamma 1 during the early induction phase of the response. It is surprising that although the generation memory is diminished, there is no quantitative difference in the development of germinal centers. Whereas entry of B cells into the memory cell pathway is dependent on CD40 ligation, the clonal expansion of the potential memory precursors in germinal centers seems not to require a CD40 signal.
MHC class I-restricted CTLs specific for antigens expressed by malignant cells are an important component of immune responses against human cancer. Recently, in melanoma a number of melanocyte differentiation antigens have been identified as potential tumor rejection antigens. In the present study, we show that by applying peptide-loaded dendritic cells, induced by granulocyte-macrophage colony-stimulating factor and interleukin 4 from peripheral blood monocytes of healthy donors, we were able to elicit melanoma-associated antigen-specific CTL in vitro. We demonstrate the induction of CTLs directed against HLA-A2.1 presented epitopes derived from tyrosinase, gp100, and Melan A/MART-1. Apart from lysis of peptide-loaded target cells, these CTLs displayed reactivity with HLA-A2.1+ melanoma tumor cell lines and cultured normal melanocytes endogenously expressing the target antigen. These data indicate that these CTLs recognize naturally processed and presented epitopes and that precursor CTLs against melanocyte differentiation antigens are present in healthy individuals. The ability to generate tumor-specific CTLs in vitro, using granulocyte-macrophage colony-stimulating factor/interleukin 4-induced dendritic cells, illustrates the potential use of this type of antigen-presenting cells for vaccination protocols in human cancer.
To better understand the events occurring during immunotherapy of liver metastases with effector cells, we have developed a clinically relevant animal model in which both effector-tumor cell interactions and survival can be evaluated. A cell line of human gastric carcinoma (HR) metastatic to the liver has been established from a patient's liver biopsy. HR cells (10 x 10(6)) injected intrasplenically metastasize into the liver of immunosuppressed nude mice, with micrometastases detectable histologically by day 4 and macrometastases by day 7. The animals subsequently develop ascites and die between days 30 and 40 after tumor injection. To investigate early metastatic events in the liver, HR cells were transduced with a plasmid containing both the lacZ gene under the control of the CMV promoter and NeoR gene. Transfectants selected for neomycin resistance were lacZ gene positive and stained blue in the presence of a beta-galactosidase substrate, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). These transfectants (HRLZ) remained lacZ gene positive for at least 25 passages in vitro. Injected intrasplenically, an HRLZ clone grew invasively in nude mice and formed liver metastases comparably to parental tumor cells. The number and localization of blue X-Gal-positive tumor cells were followed in liver tissues of animals sacrificed at various times, from 1 h to 28 days postinjection of HRLZ cells. HRLZ cells were seen in liver blood vessels and sinusoids within 1 h after injection, and the progressive growth of micrometastases and macrometastases could be followed with precision by X-Gal staining. On day 3 after injection of HRLZ cells, numerous micrometastases were established containing 12-16 tumor cells. When these 3-day established HRLZ micrometastases were treated by the intrasplenic infusion of interleukin 2 (IL2)-activated human natural killer (NK) cells selected by IL2-induced adherence to plastic (A-NK) and systemic IL2, nearly all liver micrometastases were eliminated within 24 h after a single transfer of A-NK cells (P < 0.001). This xenogeneic model was also used for adoptive immunotherapy of 7-day established liver macrometastases with human A-NK cells injected intrasplenically and exogenous IL2 given i.p. A significant decrease in the number of hepatic metastases and the weight of livers (P < 0.003) in comparison with those of control mice was observed.(ABSTRACT TRUNCATED AT 400 WORDS)
Human Langerhans cells (LC) are CD1a+ dendritic cells (DC) that function as potent antigen-presenting cells for primary and secondary immune responses. Limitations in DC/LC numbers, imposed by difficult and tedious isolation procedures, have so far precluded their use as immunogens in the generation and/or augmentation of host responses against various pathogens. Therefore, we have developed a procedure for the generation of human DC/LC from CD34+ hematopoietic progenitor cells (HPC) isolated (mean: 0.7 x 10(6)/ buffy coat and 2.6 x 10(6)/leukapheresis product) and purified ( > 95%) from the peripheral blood of healthy adults. In vitro stimulation of these cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha led to their vigorous proliferation and differentiation resulting in the emergence of CD45+/CD68+/CD3-/CD19-/CD56- leukocytes some of which (mean: 12%) express CD1a and exhibit anti-CD4 and anti-major histocompatibility complex (MHC) class II reactivity. These CD1a- leukocytes include (1) LC as evidenced by the presence of Birbeck granules (BG), (2) CD14+ monocytes, and (3) Birbeck granule-negative cells with a dendritic morphology. Addition of interleukin (IL)-4 to the cytokine cocktail interfered with the development of monocytes and led to a reduction in the overall yield but, on the other hand, resulted in an increased percentage of CD1a+ cells (mean: 24%) among all cells generated. In vitro generated CD1a+, but not CD1a- HPC-derived cells are potent stimulators of the primary mixed leukocyte reaction and, as such, promising candidates for vaccination purposes.
We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.
NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2-activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBL-derived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2-activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.
In the present study, we have analyzed the pattern of cytokines expressed by two independent dendritic cell (DC) subpopulations generated in vitro from human cord blood CD34+ progenitors cultured with granulocyte-macrophage CSF and TNF-alpha. Molecularly, we confirmed the phenotypic differences discriminating the two subsets: E-cadherin mRNA was only detected in CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were observed exclusively in CD14+-derived DC. Semiquantitative reverse-transcriptase PCR analysis revealed that both DC subpopulations spontaneously expressed IL-1alpha, IL-1beta, IL-6, IL-7, IL-12 (p35 and p40), IL-15, IL-18, TNF-alpha, TGF-beta, macrophage CSF, and granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and IFN-gamma transcripts. Both subpopulations were shown to secrete IL-12 after CD40 triggering. Interestingly, only the CD14+-derived DC secreted IL-10 after CD40 activation, strengthening the notion that the two DC subpopulations indeed represent two independent pathways of DC development. Furthermore, both DC subpopulations expressed IL-13 mRNA and protein following activation with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and IL-10, revealing the existence of different pathways for DC activation. Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by CD4+ CD11c+ CD3- DC isolated ex vivo from tonsillar germinal centers. Thus, CD14+-derived DC expressing IL-10 and factor XIIIa seemed more closely related to germinal center dendritic cellsGCDC than to Langerhans cells.
Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.
NK cell-mediated cytotoxicity is regulated by both triggering and inhibitory signals. The interaction between MHC class I molecules expressed on target cells and specific MHC class I-binding receptors expressed by NK cells generally leads to inhibition of lysis. We have shown recently that CD80 (B7-1) in mice and CD40 in humans trigger NK cell-mediated cytotoxicity in vitro. In the present study, we show that murine CD40 and CD86 (B7-2) trigger murine NK cell-mediated cytotoxicity in vitro when expressed on tumor cells. Preincubation of the transfected cell lines with anti-CD40 F(ab')2 fragments or cytolytic T lymphocyte-associated Ag-4-Ig (CTLA-4-Ig) before the cytotoxic assay abolished the triggering effect. Furthermore, radiolabeled CD40- and B7-2-expressing cells were rapidly eliminated in vivo in an NK cell-dependent manner. NK cells from CD40 ligand (CD40L)-/- or CD28-/- mice were triggered by tumor cells transfected with CD40 and B7-2, respectively, and these transfectants were rapidly eliminated in vivo when inoculated into CD40L-/- and CD28-/- mice. This suggests that the CD40 and B7-2 molecules can interact with receptors on NK cells other than CD40L and CD28, respectively, and that these may account for some of the reactivities observed in the present study. Collectively, these data demonstrate that 1) costimulatory molecules, other than B7-1, can modulate NK cell responses in vitro, 2) they can also affect NK cell-dependent responses in vivo, and 3) parts of these reactions are independent of CD28 and CD40L.
We assessed both non- and peptide-specific immune responses in prostate cancer patients before and after immunotherapy with dendritic cells exogenously pulsed with the prostate-specific membrane antigen-derived peptides, PSM-P1 and PSM-P2. For all subjects, we observed that clinical responses were strongly associated with two indicators of immunocompetence: skin test responses to recall antigens and cytokine secretion by T cells after nonspecific stimulation. In a subset of responders, we observed cytokine secretion or cytotoxicity against the immunizing peptides or an immunodominant epitope from an influenza recall antigen. The clinical results support the use of monitoring for overall immunocompetence to help determine why a patient has or has not responded to therapy. Moreover, it could be useful as an inclusion criterion to select those more likely to benefit from treatment.
To study the role of the CD40-CD40 ligand interaction in the development of memory B cells and its level of action during primary antibody responses in vivo, mice were injected with a soluble CD40 fusion protein (sCD40-gamma 1), so as to block the interaction. The effects of the treatment on the primary antibody response were reminiscent of hyper-immunoglobulin M (IgM) syndrome (HIMG1): antigen- specific IgG responses were grossly inhibited whereas the IgM response was augmented severalfold. The latter observation suggests that there is a T-dependent, CD40 ligand-independent pathway of B cell activation that leads to IgM responses and that a significant component of the IgM in HIMG1 patients is derived from T-dependent responses. The secondary response was not readily blocked by sCD40-gamma 1 treatment, indicating a relative independence of CD40 ligation of antigen-experienced B cells. The most striking finding from these studies is that the development of memory B cell populations (measured by adoptive transfer) is grossly impaired by administration of sCD40-gamma 1 during the early induction phase of the response. It is surprising that although the generation memory is diminished, there is no quantitative difference in the development of germinal centers. Whereas entry of B cells into the memory cell pathway is dependent on CD40 ligation, the clonal expansion of the potential memory precursors in germinal centers seems not to require a CD40 signal.
We have identified and purified a novel cytokine, NK cell stimulatory factor (NKSF), from the cell-free supernatant fluid of the phorbol diester-induced EBV-transformed human B lymphoblastoid cell line RPMI 8866. NKSF activity is mostly associated to a 70-kD anionic glycoprotein. The purified 70-kD protein, isolated from an SDS-PAGE gel, yields upon reduction two small species of molecular masses of 40 and 35 kD, suggesting that this cytokine is a heterodimer. When added to human PBL, purified NKSF preparations induce IFN-gamma production and synergize with rIL-2 in this activity, augment the NK cell-mediated cytotoxicity of PBL preparations against both NK-sensitive and NK-resistant target cell lines, and enhance the mitogenic response of T cells to mitogenic lectins and phorbol diesters. The three activities remain associated through different purification steps resulting in a 9,200-fold purification, and purified NKSF mediates the three biological activities at concentrations in the range of 0.1-10 pM. These data strongly suggest that the same molecule mediates these three activities, although the presence of traces of contaminant peptides even in the most purified NKSF preparations does not allow us to exclude the possibility that distinct biologically active molecules have been co-purified. The absence of other known cytokines in the purified NKSF preparations, the unusual molecular conformation of NKSF, the high specific activity of the purified protein, and the spectrum of biological activities distinguish NKSF from other previously described cytokines.
We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.
T cells recognize peptides that are bound to MHC molecules on the surface of different types of antigen-presenting cells (APC). Antigen presentation most often is studied using T cells that have undergone priming in situ, or cell lines that have been chronically stimulated in vitro. The use of primed cells provides sufficient numbers of antigen-reactive lymphocytes for experimental study. A more complete understanding of immunogenicity, however, requires that one develop systems for studying the onset of a T cell response from unprimed lymphocytes, especially in situ. Here it is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC. The dendritic cells were isolated from spleen, pulsed with protein antigens, and then administered to naive mice. Antigen-responsive T cells developed in the draining lymphoid tissue, and these T cells only recognized protein when presented on cells bearing the same MHC products as the original priming dendritic cells. In contrast, little or no priming was seen if antigen-pulsed spleen cells or peritoneal cells were injected. Since very small amounts of the foreign protein were visualized within endocytic vacuoles of antigen-pulsed dendritic cells, it is suggested that dendritic cells have a small but relevant vacuolar system for presenting antigens over a several day period in situ.
Background. Patients with testicular cancer can be cured by cisplatin-based chemotherapy in many cases. Thus, concern about possible late toxicities of treatment is warranted.
Methods. In this investigation, the absolute number of natural killer (NK) cells according to their CD56+ phenotype, NK cell activity, and antibody dependent cellular cytotoxicity (ADCC) were investigated in 29 patients with seminomas or nonseminomatous germ cell tumors (NSGCT) a median of 31 months (range, 5-73 months) after termination of chemotherapeutic treatment.
Results. No difference in the absolute number of NK cells, NK cell activity, and ADCC was found between patients with testicular cancer after either standard poly-chemotherapy consisting of bleomycin, etoposide, and cisplatin (BEP) or monotherapy with carboplatin and healthy control subjects. When patients were analyzed further using multivariate analysis, a significant (P < 0.05) detrimental influence of BEP polychemotherapy versus carboplatin monotherapy on NK cell activity was found. Moreover, NK cell activity also depended significantly (P < 0.05) on the time elapsed after chemotherapy was administered, but normalized with time. Because the absolute number of NK cells was not affected, is was assumed that polychemotherapy induced a functional defect. In a subanalysis of patients with NSGCTs, metastases at diagnosis resulted in a significant (p < 0.05) and persistent stimulation of NK cell activity but not of ADCC.
Conclusion. Cytostatic chemotherapy in patients with testicular cancer did not lead to compromised lytic effector cell function as assessed by NK cell activity and ADCC. However, a short, time-dependent reduction was found that also depended on the intensity of chemotherapeutic treatment. This finding related to the observation of a long-lasting stimulus of NK cell activity by initial metastases in patients with NSGCTs. Cancer 1995; 75:539–44.
Natural killer (NK) cells are bone marrow-derived large granular lymphocytes that express the CD56 surface antigen. The CD56bright NK subset represents approximately 10 % of all NK cells and is thought to be the least differentiated NK cell component in blood. The most mature NK cell expresses CD56 at low density and CD16 (FcRγIII) at high density, whereas CD56bright NK cells either lack CD 16 (CD56brightCD16−) or express it at low density (CD56brightCD16dim). c-kit is a tyrosine kinase receptor which is expressed on both CD34+ hemato-poietic precursor cells and CD56bright NK cells. In the current study, we characterize interleukin (IL)-2 receptor (IL-2R) and c-kit expression in each of the CD56bright subsets. Both the CD56brightCD16− and CD56brightCD16dim NK subsets express the high-affinity IL-2R and the c-kit receptor when isolated from fresh blood. However, each CD56bright NK cell subset has distinct functional responses to IL-2, the c-kit ligand (KL), or both. Activation of the high-affinity IL-2R on CD56brightCD16− NK cells induces a proliferative response that is significantly weaker than that observed in the CD56brightCD16dim NK cell subset. Incubation of the CD56brightCD16− NK cell subset with KL significantly enhances IL-2-induced proliferation, while KL has no such effect on the CD56brightCD16dim NK subset. Activation of the high-affinity IL-2R in both CD56bright subsets induces lymphokine-activated killer (LAK) activity, but the addition of KL has no effect on LAK activity. Co-stimulation of either CD56bright subset with IL-12 and concentrations of IL-2 that only saturate the high-affinity IL-2R induces substantial interferon (IFN)-γ production. The addition of KL to this co-stimulatory signal enhances IFN-γ production in both CD56bright NK subsets. The distinct functional responses to IL-2 and KL seen in the CD56brightCD16− and CD56brightCD16dim NK subsets provide insight into IL-2R signaling and suggest that each phenotype identifies a discrete stage of NK cell differentiation.
Stress and surgery have been suggested to compromise host resistance to infectious and malignant diseases in experimental and clinical settings. Because stress affects numerous physiological systems, the role of the immune system in mediating such effects is unclear. In the current study, we assessed the degree to which stress-induced alterations in natural killer (NK) cell activity underlie increased susceptibility to tumor development in F344 rats. Two stress paradigms were used: forced swim and abdominal surgery. Host resistance to tumor development was studied using 3 tumor models syngeneic to inbred F344 rats: CRNK-16 leukemia and the MADB106 mammary adenocarcinoma, both sensitive to NK activity, and the NK-insensitive C4047 colon cancer. Swim stress increased CRNK-16-associated mortality and metastatic development of MADB106 but not metastasis of C4047 cells. In both stress paradigms, stress suppressed NK activity (NKA) for a duration that paralleled its metastasis-enhancing effects on the MADB106 tumor. In vivo depletion of large granular lymphocyte/NK cells abolished the metastasis-enhancing effects of swim stress but not of surgical stress. Our findings indicate that stress-induced suppression of NKA is sufficient to cause enhanced tumor development. Under certain stressful conditions, suppression of NKA is the primary mediator of the tumor-enhancing effects of stress, while under other conditions, additional factors play a significant role. Clinical circumstances in which surgical stress may induce enhanced metastatic growth are discussed. Int. J. Cancer 80:880–888, 1999. © 1999 Wiley-Liss, Inc.
NK cells and certain CTL can recognize and lyse targets without restriction by the MHC. NK cells do not express CD3/TCR complexes and the membrane receptors participating in MHC-unrestricted cytotoxicity are largely unknown. We demonstrate that YT2C2, a human NK leukemia cell line, expresses the CD28 differentiation Ag and can spontaneously lyse both murine and human cell lines expressing B7, a B cell- activation Ag that is a ligand for CD28. The participation of CD28/B7 interactions in MHC-unrestricted cytotoxicity mediated by YT2C2 cells was demonstrated by correlation of target sensitivity with levels of B7 expression, inhibition of cytotoxicity by anti-CD28 or anti-B7 mAb, and by making both murine and human cell lines susceptible to YT2C2-mediated lysis by genetic transfection with expression vectors containing B7 cDNA. However, CD28/B7 interactions alone were insufficient to initiate cytotoxicity. mAb inhibition experiments and selection of CD54- (intercellular adhesion molecule-1) deficient B cell targets indicated that CD11a/18 (lymphocyte function-associated Ag-1) also cooperated in CD28/B7-dependent cytotoxicity. The requirement for both CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interactions in YT2C2-mediated MHC-unrestricted cytotoxicity was confirmed by demonstrating that efficient lysis of murine L cells required cotransfection with both B7 and intercellular adhesion molecule-1. These findings support the concept that MHC-unrestricted cytotoxicity may not be due to a unique receptor, but may result from interactions between an appropriate array of "adhesion" molecules with their ligands.
Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.
IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.
Dendritic cells are a system of antigen presenting cells that function to initiate several immune responses such as the sensitization of MHC-restricted T cells, the rejection of organ transplants, and the formation of T-dependent antibodies. Dendritic cells are found in many nonlymphoid tissues but can migrate via the afferent lymph or the blood stream to the T-dependent areas of lymphoid organs. In skin, the immunostimulatory function of dendritic cells is enhanced by cytokines, especially GM-CSF. After foreign proteins are administered in situ, dendritic cells are a principal reservoir of immunogen. In vitro studies indicate that dendritic cells only process proteins for a short period of time, when the rate of synthesis of MHC products and content of acidic endocytic vesicles are high. Antigen processing is selectively dampened after a day in culture, but the capacity to stimulate responses to surface bound peptides and mitogens remains strong. Dendritic cells are motile, and efficiently cluster and activate T cells that are specific for stimuli on the cell surface. High levels of MHC class-I and -II products and several adhesins, such as ICAM-1 and LFA-3, likely contribute to these functions. Therefore dendritic cells are specialized to mediate several physiologic components of immunogenicity such as the acquisition of antigens in tissues, the migration to lymphoid organs, and the identification and activation of antigen-specific T cells. The function of these presenting cells in immunologic tolerance is just beginning to be studied.
We have examined the relative alloimmunogenicity of monodisperse epidermal Langerhans cells (LC), Thy-1+ dendritic epidermal cells, and keratinocytes prepared from the skins of mice, using appropriate fluorescent-tagged mAb and flow cytometry. Graded doses of each cell type were inoculated i.v. and/or s.c. into allogeneic recipients that were selected on the basis of their degree of immunogenetic disparity with the donors of the epidermal cell (EC) inocula. From 4 to 6 wk later the spleens or draining lymph nodes of recipient mice were assayed for specific priming of cytotoxic T cells. LC proved to be extremely powerful immunogens. As few as 10 MHC-disparate EC primed allospecific T cells of mice that received i.v. or s.c. injected cells. By contrast, at least 10,000 keratinocytes were required to prime appropriate recipients, and then only when these class II MHC-negative cells were injected s.c. Thy-1 dendritic epidermal cells failed to sensitize by any route in the doses employed. With the use of appropriate donor/recipient strain combinations, it was determined that LC can effectively prime cytotoxic T cells specific for diverse types of alloantigens, including determinants encoded by class I and class II MHC genes, as well as minor histocompatibility genes. The results of these in vivo studies confirm that, among EC, the primary alloimmunogenic stimulus resides among LC, and support the hypothesis that LC play a major role in the immunogenicity of skin allografts.
CD28, a cell surface molecule expressed on all murine T cells, plays a critical role in T cell activation. We show here that NK-1.1+ cells, a subpopulation of SCID splenocytes, and IL-2-activated NK cells express CD28, although at lower levels than alpha beta T cells. Optimal proliferation of highly purified asialo GM1+ NK cells from the SCID spleen was observed in response to stimulation with IL-2 and PMA, together with anti-CD28 or L-B7+ cells. Thus, in addition to IL-2, murine NK cells require CD28-mediated costimulatory signals for optimal proliferation. IL-2-activated NK cells produced enhanced levels of IFN-gamma and TNF in response to stimulation with anti-CD28 and PMA. On the other hand, we were unable to demonstrate that CD28 signaling had any effect on NK-mediated cytotoxicity. We conclude that the CD28 costimulatory pathway is functional in NK cells and plays an important role in their proliferation and cytokine production.
Patients with testicular cancer can be cured by cisplatin-based chemotherapy in many cases. Thus, concern about possible late toxicities of treatment is warranted.
In this investigation, the absolute number of natural killer (NK) cells according to their CD56+ phenotype, NK cell activity, and antibody dependent cellular cytotoxicity (ADCC) were investigated in 29 patients with seminomas or nonseminomatous germ cell tumors (NSGCT) a median of 31 months (range, 5-73 months) after termination of chemotherapeutic treatment.
No difference in the absolute number of NK cells, NK cell activity, and ADCC was found between patients with testicular cancer after either standard polychemotherapy consisting of bleomycin, etoposide, and cisplatin (BEP) or monotherapy with carboplatin and healthy control subjects. When patients were analyzed further using multivariate analysis, a significant (P < 0.05) detrimental influence of BEP polychemotherapy versus carboplatin monotherapy on NK cell activity was found. Moreover, NK cell activity also depended significantly (P < 0.05) on the time elapsed after chemotherapy was administered, but normalized with time. Because the absolute number of NK cells was not affected, is was assumed that polychemotherapy induced a functional defect. In a subanalysis of patients with NSGCTs, metastases at diagnosis resulted in a significant (p < 0.05) and persistent stimulation of NK cell activity but not of ADCC.
Cytostatic chemotherapy in patients with testicular cancer did not lead to compromised lytic effector cell function as assessed by NK cell activity and ADCC. However, a short, time-dependent reduction was found that also depended on the intensity of chemotherapeutic treatment. This finding related to the observation of a long-lasting stimulus of NK cell activity by initial metastases in patients with NSGCTs.
Maximal T lymphocyte responses require presentation of antigen by major histocompatibility complex molecules and delivery of one or more co-stimulatory signals. Interaction of the CD28 molecule on T lymphocytes with its ligands on antigen-presenting cells (APC) initiates a critical co-stimulatory pathway inducing T lymphocyte proliferation and cytokine secretion. Dendritic cells (DC) are potent APC for a primary T lymphocyte response and potential CD28/CTLA-4 ligands on DC are, therefore, of particular functional relevance. In these experiments, the expression and function of the CD28/CTLA-4 ligands B7.1 (CD80) and B7.2 (CD86) were examined on human blood DC. Resting DC populations directly isolated by immunodepletion of lineage marker-positive cells lacked cell membrane expression of CD80 and expressed little or no CD86, although CD86, but not CD80 mRNA was detected by reverse transcription-polymerase chain reaction analysis. In contrast, low-density DC isolated after culture in vitro strongly expressed CD86 surface protein, but expressed limited or no CD80, although mRNA for both molecules were detected. Short-term culture of directly isolated DC up-regulated both CD80 and CD86 expression. Analysis of the kinetics of CD28/CTLA-4 ligand induction showed that surface CD86 was present within 8 h, whereas CD80 antigen was first detected after 24 h of culture. The functional importance of CD28/CTLA-4 ligand up-regulation on DC during T lymphocyte interactions was demonstrated by the ability of both CTLA-4Ig and CD86 monoclonal antibodies (mAb), but not CD80 mAb, to block an allogeneic mixed lymphocyte reaction stimulated by DC populations initially negative for CD80 and CD86. These results demonstrate that CD86 is both the earliest and functionally the predominant co-stimulatory CD28/CTLA-4 ligand on DC.
Dendritic cells are APCs that are unique in their potency to stimulate proliferation of primary Ag-specific responses in vitro and in vivo. In this study, we demonstrate that dendritic cells can produce IL-12, a dominant cytokine involved in the development of IFN-gamma-producing T cells. This finding resulted from our observations that dendritic cell-induced Th1 development from total CD4+ T cells upon neutralization of endogenous levels of IL-4 was IL-12-dependent. Furthermore, we demonstrate that dendritic cells can induce the development of Th1 cells from Ag-specific naive LECAM-1bright CD4+ T cells obtained from alpha beta-TCR transgenic mice, provided that CD4+ LECAM-1dull T cells, which produce significant levels of IL-4, are not present in the primary cultures. Production of IL-12 by dendritic cells was confirmed by positive immunofluoresence staining with Abs specific for the inducible IL-12 p40 subunit. This suggests that in addition to inducing proliferation and clonal expansion of naive T cells, dendritic cells, by their production of IL-12, play a direct role in the development of IFN-gamma-producing cells that are important for cell-mediated immune responses.
Tumour necrosis factor (TNF alpha) is a major inflammatory cytokine with potentiating effects on specific immune responses, including graft-versus-host disease. This study examined the contribution of TNF alpha to dendritic cell (DC)-mediated primary allogeneic T lymphocyte responses. Purified blood DC were shown to produce minimal amounts of TNF alpha mRNA but no significant TNF biological activity or secreted TNF alpha as measured by ELISA. Amplification of DC mRNA by PCR using oligonucleotide primers to CD120a (TNFRI, p55) and CD120b (TNFRII, p75) and probing with specific internal oligonucleotides, suggested that DC express the CD120b but little if any CD120a. These results were confirmed using monoclonal antibodies to the TNF receptors. Polyclonal antiserum specific for TNF alpha blocked the blood DC-stimulated allogeneic mixed leucocyte reaction (MLR). The addition of TNF alpha to suboptimal MLRs (limited DC stimulators), increased the proliferation of responding T lymphocytes. Having confirmed that T lymphocytes produce TNF alpha and express CD120b after stimulation, we sought to clarify whether the contributing effect of TNF alpha to the allogeneic MLR resulted from a TNF alpha-mediated signal stimulating DC activity, or as a result of autocrine stimulation of T lymphocytes. Pre-incubation of DC with TNF alpha did not increase DC stimulatory capacity and late addition of anti-TNF serum (up to 72 h) still had a significant inhibitory effect on the MLR. We conclude that TNF alpha is probably not involved in the initial DC-T lymphocyte interaction, but acts as an autocrine growth factor for DC induced T lymphocyte proliferation.
Human NK cells can be separated into two functionally distinct subpopulations based on the ability to rapidly respond to IL-2 by adherence to solid surfaces. To determine functions of the NK cell subsets in solid tumor tissues, adherent (A) and nonadherent (NA) NK cells were evaluated for their ability to infiltrate multicellular tumor spheroids in vitro, to kill carcinoma (CA) cell targets in these spheroids, and to mediate antitumor activity in vivo. A-NK cells were less cytolytic than NA-NK cells against CA targets in single cell suspensions or in monolayers. However, A-NK cells showed a significantly better ability than NA-NK cells to infiltrate tumor tissues and kill tumor cells in spheroids of human squamous cell CA of the head and neck or breast CA. Perilesional delivery of human A-NK cells and IL-2 resulted in regression of established human squamous cell carcinoma of the head and neck tumors growing subcutaneously in immunosuppressed nude mice. Similarly, in a xenograft model of human gastric CA metastatic to liver of nude mice, a single intrasplenic injection of A-NK cells in combination with i.p. infusions of IL-2 significantly reduced the number of established hepatic metastases (p < 0.007) and prolonged survival of the mice (p < 0.003). In contrast, NA-NK cells were ineffective in either of the in vivo xenograft tumor models. These findings demonstrate that A-NK cells represent a biologically unique and important subset of NK cells that, in contrast to the rest of NK cells, function as effector cells in solid tumor tissues and, consequently, have a great antitumor therapeutic potential.
Using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 we have established dendritic cell (DC) lines from blood mononuclear cells that maintain the antigen capturing and processing capacity characteristic of immature dendritic cells in vivo. These cells have typical dendritic morphology, express high levels of major histocompatibility complex (MHC) class I and class II molecules, CD1, Fc gamma RII, CD40, B7, CD44, and ICAM-1, and lack CD14. Cultured DCs are highly stimulatory in mixed leukocyte reaction (MLR) and are also capable of triggering cord blood naive T cells. Most strikingly, these DCs are as efficient as antigen-specific B cells in presenting tetanus toxoid (TT) to specific T cell clones. Their efficiency of antigen presentation can be further enhanced by specific antibodies via FcR-mediated antigen uptake. Incubation of these cultured DCs with tumor necrosis factor alpha (TNF-alpha) or soluble CD40 ligand (CD40L) for 24 h results in an increased surface expression of MHC class I and class II molecules, B7, and ICAM-1 and in the appearance of the CD44 exon 9 splice variant (CD44-v9); by contrast, Fc gamma RII is markedly and sometimes completely downregulated. The functional consequences of the short contact with TNF-alpha are in increased T cell stimulatory capacity in MLR, but a 10-fold decrease in presentation of soluble TT and a 100-fold decrease in presentation of TT-immunoglobulin G complexes.
This paper reviews recent publications and presents data dealing with natural killer (NK) cell activity in the tumor-infiltrating lymphocytes (TIL) and peripheral blood (PBL) of patients with solid tumors and leukemia. Cells with NK markers or function are not a prominent feature of lymphoid infiltrates in solid tumors and, when present, do not appear to correlate with other prognostic variables. Nevertheless, NK cells among IL-2-activated TIL mediate antitumor cytotoxicity. Many studies indicate that NK activity is reduced in patients with advanced cancer. In some of these studies, low NK activity has been shown to be an unfavorable prognostic variable. PBL, splenic and bone marrow NK activity in patients with acute myelogenous leukemia (AML) is also decreased. However, adherent IL-2-activated lymphocytes composed primarily of NK cells were found to be cytotoxic against AML blasts and could be generated from patients in remission and relapse. The question of whether low NK activity in solid tumors and leukemia is the result of the disease state or contributes to it, remains unanswered. Data are also presented here showing that treated, apparently disease-free patients with high PBL NK activity have a significantly longer metastasis-free survival time than those with low NK activity (n = 91, p < 0.026, Cox proportional hazards test).
T cells play a central role in the initiation and regulation of the immune response to antigen. Both the engagement of the TCR with MHC/Ag and a second signal are needed for the complete activation of the T cell. The CD28/B7 receptor/ligand system is one of the dominant costimulatory pathways. Interruption of this signaling pathway with CD28 antagonists not only results in the suppression of the immune response, but in some cases induces antigen-specific tolerance. However, the CD28/B7 system is increasingly complex due to the identification of multiple receptors and ligands with positive and negative signaling activities. This review summarizes the state of CD28/B7 immunobiology both in vitro and in vivo; summarizes the many experiments that have led to our current understanding of the participants in this complex receptor/ligand system; and illustrates the current models for CD28/B7-mediated T cell and B cell regulation. It is our hope and expectation that this review will provoke additional research that will unravel this important, yet complex, signaling pathway.
We investigated the possibility that T helper cells might enhance the stimulatory function of dendritic cells (DCs). We found that ligation of CD40 by CD40L triggers the production of extremely high levels of bioactive IL-12. Other stimuli such as microbial agents, TNF-alpha or LPS are much less effective or not at all. In addition, CD40L is the most potent stimulus in upregulating the expression of ICAM-1, CD80, and CD86 molecules on DCs. These effects of CD40 ligation result in an increased capacity of DCs to trigger proliferative responses and IFN-gamma production by T cells. These findings reveal a new role for CD40-CD40L interaction in regulating DC function and are relevant to design therapeutic strategies using cultured DCs.
NK cell-mediated cytotoxicity is influenced by triggering as well as inhibitory signals. The identification of inhibitory signals provided by MHC class I molecules has recently attracted significant attention. Much less is known about putative triggering signals. Using purified populations of mouse NK cells, we demonstrate that the CD80 (B7-1) gene product functions as a triggering signal for NK cell-mediated cytotoxicity. The strength of this response is such that it overrides the protection mediated by MHC class I molecules. Triggering of mouse NK cells by B7-1 occurred even in the absence of CD28 and could not be blocked by either anti-CD28 or anti-CTLA-4 antibodies. NK cells may thus, at least in part, use receptors other than CD28 and CTLA-4 in their interaction with B7-1. Furthermore, we demonstrate that bone marrow-derived macrophages and dendritic cells are highly susceptible to lysis by autologous NK cells.
Since either macrophages (Mphi) or dendritic cells (DC) differentiate from monocytes (MO) depending on culture conditions, we investigated the relationship of the DC and Mphi differentiation pathways. Culturing MO-enriched blood mononuclear cells with Mphi colony-stimulating factor (M-CSF) or with granulocyte/Mphi (GM)-CSF induced Mphi with a different morphology and CD14/CD1a expression. In contrast, in cultures with GM-CSF and interleukin (IL)-4, cells rapidly became nonadherent and acquired DC morphology, ultrastructure, CD1a expression, and most DC markers; they lost membrane CD14 and CD64 and capacity of phagocytosis, displayed less CD68 than Mphi, but retained nonspecific esterase activity. These DC directly developed from MO without proliferation inasmuch as only day 0 FACS-sorted MO, but not small CD14- cells, differentiated into DC when cultured with GM-CSF and IL-4, or to Mphi with M-CSF While overall cell numbers declined, DC numbers plateaued from culture day 2 onwards, indicating that most had differentiasted by then. This differentiation was radioresistant and occurred without [3H]thymidine incorporation. Commitment to differentiate into DC with GM-CSF and IL-4 was irreversible by day 2, since discontinuing IL-4 at this point did not revert cells to Mphi. Alternatively, cells rapidly converted to DC when IL-4 was added from day 2 to cultures initiated with GM-CSF only. If cultures were initiated with M-CSF and switched to GM-CSF and IL-4 after 2 or 5 days, about half of the cells still converted to DC. Thus, the capacity of MO and even of Mphi to differentiate into DC was conserved for at least this period. The increased capacity to stimulate the mixed leukocyte reaction correlated with the relative number of CD1a+ cells at any time and under each condition tested, a confirmation that these cells functionally qualify as DC. Thus, MO and even Mphi can be directed to differentiate into DC depending on the cytokine microenvironment.
Human natural killer (NK) cells are defined as being membrane CD3-, CD16+, and/or CD56+ lymphocytes; however, little is known about the ontogenic development and maturational pathways of human NK cells. The functional, phenotypic, and maturational characteristics of human umbilical cord blood (CB) NK cell subsets were studied to gain insight into the ontogenic and maturational pathways of human NK cells. We have previously shown that there is a novel subset of CD16+ CD56- NK cells present in CB. Here we further demonstrate differences in the expression of the NK-associated molecules CD2, CD7, CD8, and CD25 between CB and peripheral blood (PB) NK cells and between CB NK cell subsets. Although CB NK cell subsets were deficient in or had less lytic activity against K562 cells compared to PB NK cells, CB NK cells did possess the lytic molecules perforin and granzyme B and when artificially stimulated to secrete their granules during lytic assays, were capable of lytic activity equivalent to that of PB NK cells. Regardless of differences in phenotype and function of CB NK cell subsets, short-term and long-term incubation with cytokines induced functional (adult-like NK activity) and phenotypic (adult-like CD16+56+ or CD16-56+ surface antigen phenotype) maturation, respectively. Interleukin-2 (IL-2), IL-12, and IL-15, but not IL-7, interferon-gamma (IFN-gamma) nor tumor necrosis factor-alpha (TNF-alpha) induced functional and phenotypic maturation of CB NK cell subsets. Interestingly, culture of CB NK cell subsets with IL-2 or IL-15 led to acquisition of predominantly a CD16+56+ phenotype, while culture with IL-12 led to acquisition of both CD16+56+ and CD16-56+ phenotypes. Both functional and phenotypic maturation were not dependent upon proliferation. Studies using neutralizing anti-IFN-gamma and anti-TNF-alpha antibodies showed that survival and phenotypic maturation upon cytokine stimulation is influenced by endogenous production of TNF-alpha but not IFN-gamma. These results demonstrate that CB NK cell subsets are functionally and phenotypically immature but are capable of maturation. Additionally, CD16+56- NK cells are implicated as possible precursors of mature CD16+56+ and CD16-56+ NK cells.
Natural killer (NK) cells form a unique third group of lymphocytes that differs from T and B cells in surface phenotype, target recognition, and function. By producing cytokines and exerting cytotoxicity, NK cells participate in the resistance against microbial infections and malignant disease. The research on the molecular mechanisms of migration and target cell recognition by NK cells has recently developed rapidly. NK cells express a number of adhesion molecules common to hematopoietic lineage, bind to endothelium, extravasate, and respond to chemotactic stimuli, much resembling T cells in those respects. However, NK cells are probably capable of transmigration and infiltration merely through activation by cytokines and chemokines, as opposed to the requirement of antigen presentation in the initial activation of T cells. Target cell recognition and ensuing cytotoxicity of NK cells is a sum effect of a delicate balance between the effects of inhibitory and activating NK cell receptors. NK cells express several well-defined MHC I-recognizing receptors that inactivate their functions. In pathological alterations of MHC I expression, the inhibitory receptors do not engage and thus permit the lysis of the target cell. The receptors that trigger the cytolytic machinery of NK cells are less well known. Some candidate triggering receptors have been identified and it seems that NK cell triggering is mediated by multiple receptors, as is the inhibition of cytotoxicity. For example, NK cells clearly detect target cell-bound antibodies and thus mediate antibody-dependent cytotoxicity. They may also detect carbohydrate moieties, normal but pathologically distributed adhesion molecules, as well as ligands for a number of co-stimulatory receptors.
We classified CD56+ CD3- natural killer (NK) cells into CD2- CD56dim (CD2- NK), CD2+ CD56dim (CD2+ NK) and CD2+ CD56bright populations, and investigated mainly functional differences between the former two populations. CD2- and CD2+ NK cells were the same in their morphology and several surface molecules except for CD2. The percentages of CD2- NK cells in total NK cells were higher in the cord blood and marrow than in the peripheral blood of adults or children. Freshly isolated CD2- NK cells had CD2 in the cytoplasm, and gradually expressed it on the surface upon incubation with interleukin-2 (IL-2). These results demonstrated that CD2 is an antigen which appears on the surface during the maturation of NK cells. The granule-mediated cytotoxicities, which are mainly performed by the perforin molecule, of CD2+ NK cells against K562 and Daudi cells were higher than those of CD2- NK cells, and they were inhibited to the levels of CD2- NK cells by the addition of a blocking anti-CD2 monoclonal antibody (mAb). Fas ligand (FasL) mRNA was expressed in freshly isolated CD2+ NK cells but not in the CD2- NK cells. Neither freshly isolated NK populations showed FasL-mediated cytotoxicity, and only CD2+ NK cells lysed Fas-transfected targets after the 24-hr incubation with IL-2. Based on these results, CD2- NK cells have already developed granule-mediated cytotoxicity equal to that of CD2+ NK cells except for the CD2-associated activity, but they, unlike CD2+ NK cells, totally lack FasL-mediated cytotoxicity. These findings suggest that FasL-mediated cytotoxicity may be acquired at more mature stages of NK-cell maturation than granule-mediated cytotoxicity.
Natural killer (NK) cells are thought to play an important role in the control of metastatic dissemination. Therefore, stimulation of cytotoxic activity of NK cells against neoplastic cells could be preventive for metastatic spread. Bomirski amelanotic (Ab) melanoma of Syrian hamster is a transplantable tumor metastasizing preferably to the kidneys. During growth of the melanoma a significant depression of cytotoxic activity of NK cells of tumor hosts is observed. Treatment of melanoma-bearing hamsters with indomethacin provided in drinking water resulted in the increase of NK cytotoxic activity of blood cells and in the lower occurrence of kidney metastasis. Spleen cells obtained from healthy and melanoma-bearing hamsters were cultured in vitro with agents influencing NK activity. We found an augmentative effect of human interleukin 2 (IL2) and human tumor necrosis factor (TNF). We also observed the synergistic effect of IL2/TNF combination, which was present in both groups of animals. The stimulatory effects of cytokines could be potentiated by the additional supplementation of cultures with indomethacin. Similar experiments were performed on spleen cells isolated from the healthy and tumor-bearing animals treated in vivo with indomethacin. Also, in this group of hamsters in vitro stimulation of NK cell activity by the cytokines was effective. The studies presented may give insight into the pathogenesis of immune abnormalities seen in advanced stages of progression of Ab melanoma, and can provide an experimental basis for immunomodulation in this tumor model of spontaneous metastasis.
Stress and surgery have been suggested to compromise host resistance to infectious and malignant diseases in experimental and clinical settings. Because stress affects numerous physiological systems, the role of the immune system in mediating such effects is unclear. In the current study, we assessed the degree to which stress-induced alterations in natural killer (NK) cell activity underlie increased susceptibility to tumor development in F344 rats. Two stress paradigms were used: forced swim and abdominal surgery. Host resistance to tumor development was studied using 3 tumor models syngeneic to inbred F344 rats: CRNK-16 leukemia and the MADB106 mammary adenocarcinoma, both sensitive to NK activity, and the NK-insensitive C4047 colon cancer. Swim stress increased CRNK-16-associated mortality and metastatic development of MADB106 but not metastasis of C4047 cells. In both stress paradigms, stress suppressed NK activity (NKA) for a duration that paralleled its metastasis-enhancing effects on the MADB106 tumor. In vivo depletion of large granular lymphocyte/NK cells abolished the metastasis-enhancing effects of swim stress but not of surgical stress. Our findings indicate that stress-induced suppression of NKA is sufficient to cause enhanced tumor development. Under certain stressful conditions, suppression of NKA is the primary mediator of the tumor-enhancing effects of stress, while under other conditions, additional factors play a significant role. Clinical circumstances in which surgical stress may induce enhanced metastatic growth are discussed.
NK cells play an important role in innate immune resistance, particularly through synthesis of the pro-inflammatory cytokine IFN-gamma. This study compares the abilities of the cytokine IL-18 and the costimulatory cell surface molecule CD28 to enhance IL-12-driven IFN-gamma production by NK cells. Studies with other cytokines (IL-1beta, IL-6, TNF-alpha, IL-15) showed that IL-18 or anti-CD28 treatments were the most efficient inducers of IFN-gamma when combined with IL-12. The ability of IL-18 to enhance IFN-gamma was shown to be dependent on the presence of IL-12. Similarly, although anti-CD28 stimulation alone could enhance IFN-gamma synthesis, this effect was significantly increased in the presence of IL-12. Although neither method of costimulation required de novo protein synthesis for their effects on IFN-gamma mRNA expression, these molecules used distinct mechanisms. Specifically, nuclear run-on analysis revealed that IL-18 in combination with IL-12 enhanced the rate of transcription of the IFN-gamma gene. Conversely, treatment with anti-CD28 plus IL-12 did not significantly up-regulate the rate of transcription of the IFN-gamma gene, but stabilized IFN-gamma mRNA expression within NK cells. These findings illustrate costimulatory pathways that result in potent IFN-gamma responses by NK cells and show that although IL-18 and anti-CD28 can enhance the synthesis of IL-12-driven IFN-gamma, they employ molecular mechanisms that are distinct from one another.
Dendritic cells (DCs), antigen-presenting cells capable of priming naive T cells to specific antigens in an HLA-restricted fashion, have been demonstrated to induce protective T cell-mediated immunity in tumor-bearing animals. We performed this study to test the safety, feasibility, and clinical response of immunizations with in vitro-generated DCs, loaded with an HLA-A2-restricted peptide fragment of the tumor antigen carcinoembryonic antigen (CEA), for the treatment of patients with advanced CEA-expressing malignancies. Cell preparations enriched for autologous DCs were generated from the patients' plastic adherent peripheral blood mononuclear cells in serum-free media supplemented with granulocyte macrophage colony-stimulating factor and interleukin-4. Within the cell preparation, 66% of the cells expressed the phenotype typical for DCs (CD86high, HLA-DRhigh, and CD14low). The DCs were loaded with the CEA peptide CAP-1 and cryopreserved. Groups of three to six patients received four weekly or biweekly i.v. infusions of the CAP-1-loaded DC in escalating dose levels of 1 x 10(7), 3 x 10(7), and 1 x 10(8) cells/dose. A subset of the patients in the last group also received intradermal injections of 1 x 10(6) DCs. There were no toxicities directly referable to the treatments. One patient had a minor response, and one had stable disease. Skin punch biopsy at DC injection sites demonstrated pleomorphic infiltrates in the three patients evaluated. We conclude that it is feasible and safe to generate and administer large numbers of previously cryopreserved DCs loaded with CAP-1 peptide to patients with advanced malignancies.
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can be used for vaccination purposes, to induce a specific T-cell response in vivo against melanoma-associated antigens. We have shown that the sequential use of early-acting hematopoietic growth factors, stem cell factor, IL-3 and IL-6, followed by differentiation with IL-4 and granulocyte-macrophage colony-stimulating factor allows the in vitro generation of large numbers of immature DCs from CD34(+) peripheral blood progenitor cells. Maturation to interdigitating DCs could specifically be induced within 24 hr by addition of TNF-alpha. Here, we report on a phase I clinical vaccination trial in melanoma patients using peptide-pulsed DCs. Fourteen HLA-A1(+) or HLA-A2(+) patients received at least 4 i.v. infusions of 5 x 10(6) to 5 x 10(7) DCs pulsed with a pool of peptides including either MAGE-1, MAGE-3 (HLA-A1) or Melan-A, gp100, tyrosinase (HLA-A2), depending on the HLA haplotype. A total of 83 vaccinations were performed. Clinical side effects were mild and consisted of low-grade fever (WHO grade I-II). Clinical and immunological responses consisted of anti-tumor responses in 2 patients, increased melanoma peptide-specific delayed-type hypersensitivity reactions in 4 patients, significant expansion of Melan-A- and gp100-specific cytotoxic T lymphocytes in the peripheral blood lymphocytes of 1 patient after vaccination and development of vitiligo in another HLA-A2(+) patient. Our data indicate that the vaccination of peptide-pulsed DCs is capable of inducing clinical and systemic tumor-specific immune responses without provoking major side effects.