Anti-CD40 antibody induces antitumor and antimetastatic effects: the role of NK cells

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-gamma in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.     

Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity

NK cells possess both effector and regulatory activities that may be important during the antitumor immune response. In fact, the generation of antitumor immunity by the administration of an agonistic mAb against CD137 is NK cell-dependent. In this study, we report that NK cells could be induced by IL-2 and IL-15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-gamma secretion, but not their cytolytic activity. Importantly, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or "helper" activity for CD8(+)CTL. Furthermore, tumor-specific CTL activity against P815 tumor Ags was abrogated following anti-CD137 treatment in NK-depleted mice. We further demonstrate that CD137-stimulated helper NK cells expressed the high-affinity IL-2R and were hyperresponsive to IL-2. Taken together with previous findings that CD137 is a critical receptor for costimulation of T cells, our findings suggest that CD137 is a stimulatory receptor for NK cells involved in the crosstalk between innate and adaptive immunity.     

Intradermal delivery of IL-12 naked DNA induces systemic NK cell activation and Th1 response in vivo that is independent of endogenous IL-12 production.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-gamma and IFN-gamma-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-gamma proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o- plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o- to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o- into BALB/c IL-12 p40-/- mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-gamma protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.     

Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro

We recently reported that the direct antitumor effectors in the liver induced by alpha-galactosylceramide (alpha-GalCer) are NK cells that are activated by the IFN-gamma produced from NK1.1 Ag(+) T cells (NKT cells) specifically stimulated with alpha-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the alpha-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-gamma and IL-4 concentrations as well as alanine aminotransferase levels after the alpha-GalCer injection increased in an age-dependent manner. An alpha-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of alpha-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-gamma and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1(+) cells. Furthermore, liver mononuclear cells from old mice stimulated with alpha-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most alpha-GalCer-injected old mice, but no young mice, died, while anti-IFN-gamma Ab pretreatment completely inhibited mouse mortality. However, alpha-GalCer-induced hepatic injury did not improve at all by anti-IFN-gamma Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-gamma is responsible for alpha-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-gamma.     

Systemic administration of LPD prepared with CpG oligonucleotides inhibits the growth of established pulmonary metastases by stimulating innate and acquired antitumor immune responses

Intravenous (i.v.) administration of a lipopolyplex consisting of a ternary complex of DOTAP:cholesterol cationic liposomes, protamine sulfate, and noncoding plasmid DNA (LPD-pDNA) is capable of stimulating a potent Th-1 cytokine response and inhibiting the growth of established tumors in mice. Both activities are mainly elicited by unmethylated CpG motifs in the plasmid DNA (pDNA) component, which are bacterial in origin. Since oligodeoxynucleotides (ODN) that possess a consensus immunostimulatory CpG motif of RRCpGYY (R is purine and Y is pyrimidine) can mimic the immunostimulatory actions of bacterial DNA, we hypothesized that i.v. administration of LPD prepared with GpG-ODN would mimic the ability of LPD-pDNA to stimulate Th-1 cytokines and antitumor activity and provide an improved vector for probing the immune mechanisms underlying the observed antitumor effects. These hypotheses were tested for the treatment of established 24JK experimental pulmonary metastases that are syngeneic in C57BL/6 mice. Mice treated with LPD containing 25 microg of the prototypical phosphodiester (PO) CpG-ODN 1668 (tccatGACGTTcctgatgct, motif capitalized) demonstrated a dramatic reduction in lung tumor burden (>80% inhibition, P<0.01) compared to dextrose-treated controls. The antitumor effect was dependent on the CpG dinulceotide and correlated with the ability to stimulate serum Th-1 cytokines (TNF-alpha, IL-12, and IFN-gamma). Both activities required assembly of CpG-ODN in a cationic liposome/DNA complex (lipoplex) or the LPD lipopolyplex. LPD delivery of both PO-1668 and phosphorothioated (PS)-1668 stimulated a greater cytokine response compared to delivery of free ODN. Furthermore, within the LPD complex, both PO- and PS-1668 had similar ability to stimulate Th-1 cytokines with respect to potency and duration of response, thus eliminating the need for the PS modification. In tumor cell lysis assays, LPD-CpG DNA stimulated development of an acquired, tumor-specific CD8+ cytotoxic T-lymphocyte (CTL) activity that was dependent on CpG DNA. LPD was also capable of stimulating NK activity; however, this was not dependent on CpG DNA. Only formulations that concomitantly stimulated NK activity and CpG-specific, Th-1 cytokine were capable of stimulating the development of tumor-specific CTL activity and significant inhibition of tumor growth. Thus, we propose a model where CpG DNA in complex with cationic liposome-based lipoplexes or lipopolyplexes stimulates antitumor NK activity and CpG-stimulated Th-1 cytokine production. The combination of these two activities of the innate immune system subsequently direct the development of an acquired, tumor-specific CTL response that in total are effective for inhibiting the growth of established tumors in mice.     

Loss of IFN-gamma production by invariant NK T cells in advanced cancer

Invariant NK T cells express certain NK cell receptors and an invariant TCRalpha chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-gamma, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (alpha-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-gamma and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-gamma production and a striking decrease in their IFN-gamma:IL-4 ratio. The IFN-gamma deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-gamma and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.     

IL-28 elicits antitumor responses against murine fibrosarcoma

IL-28 is a recently described antiviral cytokine. In this study, we investigated the biological effects of IL-28 on tumor growth to evaluate its antitumor activity. IL-28 or retroviral transduction of the IL-28 gene into MCA205 cells did not affect in vitro growth, whereas in vivo growth of MCA205IL-28 was markedly suppressed along with survival advantages when compared with that of controls. When the metastatic ability of IL-28-secreting MCA205 cells was compared with that of controls, the expression of IL-28 resulted in a potent inhibition of metastases formation in the lungs. IL-28-mediated suppression of tumor growth was mostly abolished in irradiated mice, indicating that irradiation-sensitive cells, presumably immune cells, are primarily involved in the IL-28-induced suppression of tumor growth. In vivo cell depletion experiments displayed that polymorphonuclear neutrophils, NK cells, and CD8 T cells, but not CD4 T cells, play an equal role in the IL-28-mediated inhibition of in vivo tumor growth. Consistent with these findings, inoculation of MCA205IL-28 into mice evoked enhanced IFN-gamma production and cytotoxic T cell activity in spleen cells. Antitumor action of IL-28 is partially dependent on IFN-gamma and is independent of IL-12, IL-17, and IL-23. IL-28 increased the total number of splenic NK cells in SCID mice and enhanced IL-12-induced IFN-gamma production in vivo and expanded spleen cells in C57BL/6 mice. Moreover, IL-12 augmented IL-28-mediated antitumor activity in the presence or absence of IFN-gamma. These findings indicate that IL-28 has bioactivities that induce innate and adaptive immune responses against tumors.     

Mechanisms of the antimetastatic effect in the liver and of the hepatocyte injury induced by alpha-galactosylceramide in mice

The role of mouse liver NK1.1 Ag(+) T (NKT) cells in the antitumor effect of alpha-galactosylceramide (alpha-GalCer) has been unclear. We now show that, whereas alpha-GalCer increased the serum IFN-gamma concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of alpha-GALCER: Injection of Abs to IFN-gamma also inhibited the alpha-GalCer-induced increase in antitumor cytotoxicity of MNCS: alpha-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas alpha-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCS: The alpha-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-gamma. Depletion of CD8(+) T cells did not affect the alpha-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of alpha-GalCer on the survival of B6 mice. Thus, IFN-gamma produced by alpha-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8(+) T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate alpha-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.     

Oral administration of high molecular mass poly-gamma-glutamate induces NK cell-mediated antitumor immunity

We analyzed the in vivo tumor regression activity of high molecular mass poly-gamma-glutamate (gamma-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa gamma-PGA or beta-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-gamma secretion in mice treated with higher molecular mass gamma-PGA (2000 kDa) vs those treated with lower molecular mass gamma-PGA (10 or 100 kDa) or beta-glucan. We then examined the effect of oral administration of 10- or 2000-kDa gamma-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass gamma-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that gamma-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-gamma knockout mice. Taken together, we demonstrated that oral administration of high molecular mass gamma-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.     

Systemic administration of IL-23 induces potent antitumor immunity primarily mediated through Th1-type response in association with the endogenously expressed IL-12

IL-23, a cytokine, which is composed of the p40 subunit shared with IL-12 and the IL-23-specific p19 subunit, has been shown to preferentially act on Th1 effector/memory CD4+ T cells and to induce their proliferation and IFN-gamma production. The IL-23 is also reported to act on Th17-CD4+ T cells, which are involved in inducing tissue injury. In this study, we examined the antitumor effects associated with systemic administration of IL-23 and their mechanisms in mouse tumor system. Systemic administration of high-dose IL-23 was achieved using in vivo electroporation of IL-23 plasmid DNA into the pretibial muscles of C57BL/6 mice. The IL-23 treatment was associated with significant suppression of the growth of pre-existing MCA205 fibrosarcoma and prolongation of the survival of treated mice without significant toxicity when compared with those of the mice treated with EGFP. Although the therapeutic outcomes were similar to those with the IL-12 treatment, the IL-23 treatment induced characteristic immune responses distinctive to those of IL-12 treatment. The IL-23 administration even at the therapeutic levels did not induce detectable IFN-gamma concentration in the serum. In vivo depletion of CD4+ T cells, CD8+ T cells, or NK cells significantly inhibited the antitumor effects of IL-23. Furthermore, the CD4+ T cells in the lymph nodes in the IL-23-treated mice showed significant IFN-gamma and IL-17 response upon anti-CD3 mAb stimulation in vitro. These results and the ones in the IFN-gamma or IL-12 gene knockout mice suggest that potent antitumor effects of IL-23 treatment could be achieved when the Th1-type response is fully promoted in the presence of endogenously expressed IL-12.     

Trafficking and activation of murine natural killer cells: differing roles for IFN-gamma and IL-2

The repeated ip injection of highly purified recombinant IFN-gamma or IL-2 resulted in a local increase in peritoneal NK activity. This increase in lytic activity was paralleled by increases in the number of peritoneal leukocytes reacting with a rat monoclonal antibody directed against the NK cell-associated surface antigen LGL-1. LGL-1 reacts specifically with the majority of murine NK cells in BALB/c and C57BL/6 mice. A single injection of IFN-gamma induced more peritoneal NK activity at 24 hr than IL-2 on a protein basis. Both cytokines induced increases in the number of LGL-1+ peritoneal cells by 24 hr after injection. Simultaneous injection of suboptimal amounts of IFN-gamma (100 U) and IL-2 (10,000 U) resulted in a significant augmentation of peritoneal NK activity over that observed with either cytokine alone. Also, the peritoneal NK activity generated in response to ip injection of high doses of IL-2 (100,000 U) could be dramatically reduced by simultaneous injection of a neutralizing monoclonal antibody to IFN-gamma. Administration of IFN-gamma 1 day prior to IL-2 resulted in a significant augmentation of the NK activity above that observed with the individual cytokines. In contrast, injection of IL-2 prior to IFN-gamma did not enhance NK activity over that observed with the individual cytokines. Both cytokines must be injected ip for the complementary effects of IFN-gamma and IL-2 on peritoneal NK activity to occur. In contrast, in vitro incubation of peritoneal leukocytes with IFN-gamma resulted in neither a significant enhancement of NK lytic activity nor an increase in the number of LGL-1+ cells. In vitro treatment of peritoneal leukocytes with IL-2 always resulted in significant augmentation of NK lytic activity in the absence of any increase in the number of LGL-1+ cells. These data are consistent with the hypothesis that the local release of IFN-gamma increases peritoneal NK activity by promoting the influx of blood-borne LGL-1+ NK cells from other sites. In contrast, low doses of IL-2 augment the lytic activity of local resident NK cells, whereas high doses of this cytokine induce both an activation of local NK cells and emigration of LGL-1+ NK cells from other sites due to the endogenous generation of IFN-gamma within the peritoneal cavity. Therefore, the local release of IFN-gamma may play an important role in regulating NK cell infiltration in vivo.     

Dendritic cell-induced activation of adaptive and innate antitumor immunity

While studying Ag-pulsed syngeneic dendritic cell (DC) immunization, we discovered that surprisingly, unpulsed DCs induced protection against tumor lung metastases resulting from i.v. injection of a syngeneic BALB/c colon carcinoma CT26 or a syngeneic C57BL/6 lung carcinoma LL/2. Splenocytes or immature splenic DCs did not protect. The protection was mediated by NK cells, in that it was abrogated by treatment with anti-asialo-GM1 but not anti-CD8, and was induced by CD1(-/-) DCs unable to stimulate NKT cells, but did not occur in beige mice lacking NK cells. Protection correlated with increased NK activity, and increased infiltration of NK but not CD8(+) cells in lungs of tumor-bearing mice. Protection depended on the presence of costimulatory molecules CD80, CD86, and CD40 on the DCs, but surprisingly did not require DCs that could make IL-12 or IL-15. Unexpectedly, protection sensitive to anti-asialo-GM1 and increased NK activity were still present 14 mo after DC injection. As NK cells lack memory, we found by depletion that CD4(+) not CD8(+) T cells were required for induction of the NK antitumor response. The role of DCs and CD4(+) T cells provides a novel mechanism for NK cell induction and innate immunity against cancer that may have potential in preventing clinical metastases.     

Interleukin-12 activates NK cells for IFN-gamma-dependent and NKT cells for IFN-gamma-independent antimetastatic activity

Mechanisms involved in the antimetastatic effect of IL-12 were analyzed in a mouse model of experimental metastasis with either syngeneic fibrosarcoma cells colonizing the lungs or syngeneic B cell lymphoma cells colonizing the liver. IL-12 pretreatment effectively reduced the number of tumor colonies in both systems. This effect was already manifest 24 hours after tumor cell injection, indicating a T and B cell-independent mechanism. Therefore, the involvement of NK and alphabetaNKT cells was investigated using mice with defective NK and alphabetaNKT cell functions. Mice with impaired NK functions due to NK cell depletion, were less responsive to the antimetastatic IL-12 effect. IL-12 treatment failed to inhibit metastasis in beta2-microglobulin-deficient mice which lack alphabetaNKT cells in addition to having impaired NK cell activity, thus, demonstrating the functional importance of IL-12-activated NK and alphabetaNKT cells. While the IL-12-induced antimetastatic effect of NK cells was dependent on IFN-gamma action, IL-12 activation of alphabetaNKT cells did not involve IFN-gamma. The neutralization of IFN-gamma or the use of IFN-gamma receptor-deficient mice did not alter the IL-12-induced effect in the absence of NK cells. Activation of effector cells of the innate immune system, such as NK and alphabetaNKT cells, seems to be the main mechanism for the antimetastatic effect of IL-12.     

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.     

A novel dendritic cell subset involved in tumor immunosurveillance

The interferon (IFN)-gamma-induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-gamma is not the conventional NK cell but a subset of B220(+)Ly6C(-) dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220(+)NK1.1(+) dendritic cells secrete high levels of IFN-gamma and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2(-/-)Il2rg(-/-) mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response.     

Essential role of bystander cytotoxic CD122+CD8+ T cells for the antitumor immunity induced in the liver of mice by alpha-galactosylceramide

We recently reported that NK cells and CD8(+) T cells contribute to the antimetastatic effect in the liver induced by alpha-galactosylceramide (alpha-GalCer). In the present study, we further investigated how CD8(+) T cells contribute to the antimetastatic effect induced by alpha-GalCer. The injection of anti-CD8 Ab into mice 3 days before alpha-GalCer injection (2 days before intrasplenic injection of B16 tumors) did not inhibit IFN-gamma production nor did it reduce the NK activity of liver mononuclear cells after alpha-GalCer stimulation. However, it did cause a reduction in the proliferation of liver mononuclear cells and mouse survival time. Furthermore, although the depletion of NK and NKT cells (by anti-NK1.1 Ab) 2 days after alpha-GalCer injection no longer decreased the survival rate of B16 tumor-injected mice, the depletion of CD8(+) T cells did. CD122(+)CD8(+) T cells in the liver increased after alpha-GalCer injection, and antitumor cytotoxicity of CD8(+) T cells in the liver gradually increased until day 6. These CD8(+) T cells exhibited an antitumor cytotoxicity toward not only B16 cells, but also EL-4 cells, and their cytotoxicity significantly decreased by the depletion of CD122(+)CD8(+) T cells. The critical, but bystander role of CD122(+)CD8(+) T cells was further confirmed by adoptive transfer experiments into CD8(+) T cell-depleted mice. Furthermore, it took 14 days after the first intrasplenic B16/alpha-GalCer injection for the mice to generate CD8(+) T cells that can reject s.c. rechallenged B16 cells. These findings suggest that alpha-GalCer activates bystander antitumor CD122(+)CD8(+) T cells following NK cells and further induces an adaptive antitumor immunity due to tumor-specific memory CD8(+) CTLs.     

Impairment of natural killer (NK) cells is an important factor in a weak Th1-like response in irradiated mice

In whole-body-irradiated (WBI) mice, levels of the canonical Th1 cytokine IFN-gamma (IFNG) have been shown to be markedly reduced, resulting in a Th1/Th2 imbalance. In this study, the influence of natural killer (NK) cells on the balance of this Th1/Th2 immune response was evaluated in WBI mice. Although NK cells are one of the types of cells that secreteIFN-gamma, NK cell activity tends to be minimal, even at 7 weeks after irradiation. In NK cell-depleted mice, the levels of Th1-related cytokines were lower than those of the control mice and were correlated with lower IgG2a production and elevated IgE and IgG1 production. These results indicated that NK cells have a crucial role in the final differentiation of Th cells into Th1 cells. The impairment of NK cells in the WBI mice was confirmed by the observation that NK cells from the WBI mice induced a decrease in the generation of IFN-gamma by the NK cell-depleted spleen lymphocytes from normal mice. Also, the WBI mice that received NK cells obtained from the normal mice generated more IgG2a, IL12 and IFN-gamma. Our results indicate that the impairment of NK cells is an important factor in the reduced Th1-like response in irradiated mice.     

CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.