Effect of surgical stress on murine natural killer cell cytotoxicity

Natural killer cell cytotoxicity (NKCC) against tumors may be important in preventing in vivo solid tumor dissemination. Multiple animal models demonstrate increased rates of tumor dissemination after surgical stress; previously, we have observed that surgical stress impairs murine NKCC. Because of the importance of surgery in the control of solid tumors, it appeared valuable to examine the mechanism underlying surgical stress impairment of NKCC. The results of this study demonstrate that postsurgical suppression of NKCC begins as early as 2 hr after murine hind limb amputation, reaches nadir at 4 days, and does not recover to control level until postoperative day 12. Anesthetic treatment alone does not cause comparable NKCC suppression. The suppression of NKCC accompanied changes in both splenic size and morphology. The immune suppression was observed in multiple compartments including peripheral blood, bone marrow, and spleen. Mixing experiments demonstrated that surgical stress per se generated a suppressor cell population affecting NKCC. The observed suppression apparently required cell-to-cell contact, because supernatants from 4 and 18 hr cultures of suppressor cells did not cause suppression. The observed suppression was prevented by perioperative treatment with the pyrimidinone analog 2-amino-5-bromo-6-phenyl-4-pyrimidinol. These preclinical observations point to the future prospect of NK-specific perioperative immunotherapy that may help prevent possible tumor dissemination from occurring at the time of surgery.     

A poxvirus protein that binds to and inactivates IL-18, and inhibits NK cell response

IL-18 induces IFN-gamma and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R. The dissociation constant of p13 for murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectromelia evades the host immune response.     

Perioperative IFN-alpha to avoid surgically induced immune suppression in colorectal cancer patients

Surgical treatment of colorectal cancer is associated with postoperative immunosuppression, which might facilitate dissemination of tumor cells and outgrowth of minimal residual disease/(micro) metastases. Minimal residual disease has been shown to be of prognostic relevance in colorectal cancer. Therefore, stimulation of (anti-tumor) immune responses may be beneficial in the prevention of metastases formation. Important anti-tumor effector cells, which serve this function, are natural killer (NK) cells, CD8+ lymphocytes (CTL), dendritic cells (DC) and macrophages. In this review the immunomodulating properties of IFN-alpha are discussed, with a particular focus on perioperative stimulation of immune function in cancer patients. IFN-alpha is known to enhance innate immune functions such as stimulation of NK cells, transition from innate to adaptive responses (activation of DC) and regulating of CD8+ CTL activity and memory. Moreover, it exerts direct antitumor effects by regulating apoptosis and cell cycle. In several clinical trials, perioperative administration of IFN-alpha has indeed been shown to improve T cell responsiveness, prevent impairment of NK cell cytotoxicity and increase expression of activation markers on NK, T and NKT cells. In a clinical pilot study we showed in colorectal cancer patients that received perioperative IFN-alpha enhanced activation markers on T cells and NK cells, combined with better-preserved T cell function as indicated by phytohemaggluttinin skin tests. In the liver of these patients significantly more CD8+ T cells were found. In conclusion, IFN-alpha provides an effective adjuvant in several forms of cancer and improves several postoperative immune functions in perioperative administration. However, larger clinical trials are necessary to investigate effects on disease-free and overall survival.     

Curcumin reverses breast tumor exosomes mediated immune suppression of NK cell tumor cytotoxicity

An important characteristic of tumors is that they at some point in their development overcome the surveillance of the immune system. Tumors secrete exosomes, multivesicular bodies containing a distinct set of proteins that can fuse with cells of the circulating immune system. Purified exosomes from TS/A breast cancer cells, but not non-exosomal fractions, inhibit (at concentrations of nanograms per ml protein) IL-2-induced natural killer (NK) cell cytotoxicity. The dietary polyphenol, curcumin (diferuloylmethane), partially reverses tumor exosome-mediated inhibition of natural killer cell activation, which is mediated through the impairment of the ubiquitin-proteasome system. Exposure of mouse breast tumor cells to curcumin causes a dose-dependent increase in ubiquitinated exosomal proteins compared to those in untreated TS/A breast tumor cells. Furthermore, exosomes isolated from tumor cells pretreated with curcumin have a much attenuated inhibition of IL-2 stimulated NK cell activation. Jak3-mediated activation of Stat5 is required for tumor cytotoxicity of IL-2 stimulated NK cells. TS/A tumor exosomes strongly inhibit activation of Stat5, whereas the tumor exosomes isolated from curcumin-pretreated tumor cells have a lowered potency for inhibition of IL-2 stimulated NK cell cytotoxicity. These data suggest that partial reversal of tumor exosome-mediated inhibition of NK cell tumor cytotoxicity may account for the anti-cancer properties of curcumin.     

Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

Natural killer cell cytotoxicity: role of calmodulin

The phenothiazine derivatives, fluphenazine and trifluoperazine which are known to bind to calmodulin and to inhibit its activity, abrogate the development of both spontaneous and interferon-enhanced cytotoxicity of mouse splenic lymphocytes enriched for NK cell activity. Phenothiazines also inhibit the rapid increase in cyclic GMP levels in interferon-treated splenic lymphocytes. Furthermore, treatment of mouse splenic lymphocytes with electrophoretically pure interferon, alpha/beta caused a marked decrease in the level of calmodulin within 1 to 4 hours. These results provide evidence that calmodulin may play a role in the development of NK cell cytotoxicity and that the effect of interferon on calmodulin may constitute part of the molecular mechanism of interferon action.     

NK cell adoptive transfer combined with Ontak-mediated regulatory T cell elimination induces effective adaptive antitumor immune responses

Previous work from our laboratory showed that hydrocortisone (HC) combined with IL-15 induces expansion of activated human NK cells. We set up an experimental tumor model to evaluate the use of adoptively transferred, HC plus IL-15 (HC/IL-15)-activated and -expanded murine NK cells in the treatment of syngeneic mice carrying established lung metastases of the CT26 transplantable tumor. We also examined the effect of denileukin diftitox (Ontak) on the depletion of regulatory T cells to enhance the in vivo antitumor immunity induced by the adoptively transferred NK cells. Our results clearly demonstrate that murine DX5(+) NK cells are largely expanded in the presence of IL-15 plus HC while retaining intact their functional status. Moreover, when intravenously infused, they mediated significant antitumor responses against CT26 lung tumors in syngeneic BALB/c animals that were further enhanced upon pretreatment of the tumor-bearing animals with Ontak. Total splenocytes and isolated splenic T cells from NK-treated mice responded in vitro against CT26 tumor cells as evidenced by IFN-γ-based ELISPOT, proliferation, and cytotoxicity assays. Importantly, animals treated with Ontak plus adoptive transfer of HC/IL-15-expanded NK cells significantly retarded CT26 tumor growth after a rechallenge with the same tumor s.c. in their flanks. Taken altogether, our data suggest that NK cell adoptive transfer can trigger adaptive antitumor T cell responses, and regulatory T cell depletion by Ontak is mandatory for enabling HC/IL-15-activated NK cells to promote long-lasting adaptive antitumor immunity.     

Effects of recombinant murine tumor necrosis factor-alpha on immune function

TNF-alpha is a macrophage-derived cytokine with diverse biologic activities, including potent immunomodulatory effects. In vitro studies have implied that TNF-alpha has predominantly proinflammatory and immunostimulatory effects, but paradoxically in vivo studies have demonstrated that administration of TNF-alpha suppresses murine lupus. To assess the effects of TNF-alpha on immune function in normal mice, we treated C57BL/6 mice with recombinant murine TNF-alpha (10 micrograms i.p.) or PBS on alternate days for up to 8 wk. Administration of TNF-alpha decreased the percentage of splenic T and B cells and increased the percentage of splenic macrophages without significantly altering the total number of mononuclear cells. Administration of TNF-alpha also caused progressive inhibition of splenic lymphocyte function, out of proportion to the quantitative reduction in B and T cells. After 8 wk of therapy, the proliferative responses of splenic lymphocytes to Con A, PHA, and LPS were reduced by 100, 90, and 60%, respectively, in treated mice compared with control mice. The reduction in T cell proliferation was due primarily to alteration of accessory cell function rather than direct inhibition of T cell function. Treatment with TNF-alpha markedly inhibited T cell cytotoxicity induced by immunization with allogenic target cells, and it virtually ablated NK cell activity. Inhibition of these in vitro tests of lymphocyte function correlated with inhibition of delayed type hypersensitivity in vivo. In contrast, treatment with TNF-alpha did not impair humoral immunity. These findings imply that TNF-alpha may affect cell-mediated immunity more profoundly than humoral immunity. This observation may be relevant to the mechanism whereby TNF-alpha suppresses murine lupus.     

Phospholipase C-gamma 2 is a critical signaling mediator for murine NK cell activating receptors

Phospholipase C-gamma (PLCgamma) is a key regulator of intracellular Ca(2+) mobilization. Two isoforms of PLCgamma have been identified, PLCgamma1 and PLCgamma2. Previously, in vitro studies indicated that activating NK cell receptors signal through both isoforms. However, PLCgamma2 deficiency alone was sufficient to induce a substantial impairment of NK cell-mediated cytotoxicity in vitro. Why PLCgamma2 is more important than PLCgamma1 for NK cell activation and whether PLCgamma2 is also critical for NK cell development, secretion of IFN-gamma, and clearance of viral infections in vivo is not known. In this study, we report that PLCgamma2 is the predominant isoform expressed in murine NK cells. PLCgamma2 deficiency did not affect NK cell numbers in bone marrow and spleen, but acquisition of Ly49 receptors by NK cells was partially impaired. PLCgamma2-deficient NK cells exhibited a dramatic impairment of cytolytic function and IFN-gamma production upon ligation of activating receptors, whereas they did secrete IFN-gamma in response to cytokines. Consequently, mice lacking PLCgamma2 controlled murine CMV infection substantially less effectively than did wild-type animals, and this defect was most evident in the spleen, where viral clearance mostly depends on NK cell lytic function. These results demonstrate that PLCgamma2 is crucial for development of the NK cell receptor repertoire and signaling of activating NK cell receptors, mediating optimal NK cell function in vivo.     

In vitro modulation of mouse natural killer (NK) cell activity by the serum thymic factor (FTS)

Previous studies indicated that the serum thymic factor (FTS) could modulate in vivo the level of splenic natural killer (NK) cell activity in mice. The present report shows that such an effect is also observed after a short term in vitro incubation of the effector cells with FTS. The regulatory effects of FTS result in an increase or a decrease of the splenic NK cell cytotoxicity depending upon the age and the mouse strain. Furthermore, FTS is able to enhance the NK cell activity of thymus and bone marrow cells which are known to be weakly reactive in NK cytotoxicity. Depletion experiments demonstrated that the FTS-induced increase of NK cell activity was not mediated by Thy 1+ cells nor macrophages, thus suggesting a direct action of FTS on the effector cells. Comparative studies using other thymic hormones revealed similar patterns of reactivity. These results favor the hypothesis of a close relationship between the thymus and NK cells.     

A novel negative regulator molecule, Cho-1, is involved in the cytotoxicity by human natural killer cells but not in cytotoxic T lymphocytes.

We previously reported the cytotoxic negative regulatory molecule, Cho-1, that was expressed on the cell surface of rat fetal fibroblast cells in the cytotoxicity by natural killer (NK) cells. This molecule was IFN-gamma-inducible, but appeared to be different from MHC class I. It was expressed on NK-resistant cells but not on NK-sensitive murine target cells such as YAC-1. In this paper, first we determined whether Cho-1 could also act as the negative regulatory molecule in a human NK-resistant HEPM line. Our data strongly suggested that Cho-1 could act as such a negative regulatory molecule in human NK cytotoxicity. The immunoprecipitates made with HEPM cell lysate and anti-MHC class I monoclonal antibody (mAb) did not react against anti-Cho-1 mAb, indicating that Cho-I was different from MHC class I. Second, an assessment was made as to whether or not this molecule is involved in the cytotoxicity of CD8 (+) cytotoxic T lymphocytes (CTL) against human autologous tumor cells. The data indicated that although this cell surface molecule was expressed on certain tumor lines, it was not involved in the cytotoxic mechanism of CTL. Thus, Cho-1 appeared to be the novel regulatory molecule in the NK cytotoxic mechanism.     

Inducible costimulator costimulates cytotoxic activity and IFN-gamma production in activated murine NK cells

The functions of NK cells are regulated by the balance of activating and inhibitory signals. The inhibitory NK cell receptors are well understood; however, less is known about the activating signaling pathways. To explore whether a costimulatory receptor, inducible costimulator (ICOS), is involved in NK cell function, we assessed the role of ICOS in NK cell-mediated cytotoxicity and cytokine production. In addition, to determine whether ICOS contributes to the elimination of tumors in vivo, we examined the tumor growth survival of mice injected with a tumor expressing the ICOS ligand, B7RP-1. We found that ICOS was up-regulated by cytokine stimulation in murine NK cells. Consistent with ICOS expression on activated NK cells, ICOS-dependent cytotoxicity and IFN-gamma production were observed, and appeared to require signaling through the phosphoinositide 3-kinase pathway. Interestingly, ICOS-mediated stimulation allowed activated NK cells to kill more efficiently tumor cells expressing MHC class I. Furthermore, fewer metastases appeared in the liver and spleen of mice injected with the ICOS ligand-expressing tumor compared with mice bearing the parental tumor. These results indicate that NK cell functions are regulated by ICOS.     

Functional and biochemical analysis of CD16 antigen on natural killer cells and granulocytes

CD16 Ag is associated with the low affinity FcR for IgG expressed on human NK cells and granulocytes. In this study, we demonstrate that NK cells specifically lyse murine anti-CD16 hybridoma cell lines, but do not lyse hybridomas against other cell surface differentiation Ag expressed on NK cells. Moreover, the CD18 structure is involved in the CD16-specific xenogeneic interaction between human effector cells and murine hybridoma target cells. Although interaction with anti-CD16 hybridomas or antibodies triggers the cytolytic mechanism of NK cells, this interaction does not induce cellular proliferation. In contrast to NK cells, CD16+ granulocytes do not lyse anti-CD16 hybridoma cell targets and do not mediate ADCC against antibody-coated human tumor cell targets. These findings indicate a fundamental difference in the antibody-dependent cellular cytotoxicity mechanisms of NK cells and granulocytes. Comparative biochemical analysis of CD16 on NK cells and granulocytes revealed significant differences in the size of the polypeptides obtained after removal of N-linked carbohydrate residues with endo-F and N-glycanase digestion.     

Recognition of virus infected cells by NK cells

NK cells show cytotoxicity against virus-infected cells and tumor cells and play an important role in host defense. Although mecheanism of target cell recognition by NK cells have been unclear for a long time, it has recently been elucidated that certain NK cell receptors specifically recognize virus products. Furthermore, expression pattern of NK cell receptors, which consist of activating and inhibitory receptors, determines susceptibility to virus-infection. Here, we review recent progress of mechanism of recognition of virus-infected by NK cells.     

CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.     

Alternative mechanisms of natural killer cell activation during herpes simplex virus infection

Although NK cells can kill both malignant cells and virus-infected cells without prior sensitization, it has remained unclear whether the mechanism by which an NK cell is activated in the presence of a tumor cell is similar to that induced by the presence of a virus-infected cell. In our experimental system using homogeneous populations of cloned human CD16+ NK cells, we found that HSV-infected target cells do not induce in the NK cells the same pharmacologically-active second messengers elicited by NK-sensitive tumor cells. Although phosphoinositide turnover and calcium signaling were generated in NK cells exposed to NK-sensitive tumor cells, the recognition of HSV-infected cells by NK cells did not result in similar transmembrane signaling. Furthermore, depending on the cell type infected by HSV, alternative mechanisms of cytotoxicity were employed. HSV-infected foreskin fibroblasts were rapidly and selectively killed by cloned NK cells without a requirement for IFN or accessory cells. In contrast to this direct cytotoxicity against HSV-infected foreskin fibroblasts, NK cell-mediated cytotoxicity against an HSV-infected fibrosarcoma cell line (1591) was dependent on IFN-alpha production by accessory cells. Importantly, in both systems of cytotoxicity, IFN-alpha activation of NK cells resulted in augmented killing against both infected and uninfected targets. These results suggest that NK cell activation induced during antiviral immunity is distinct from activation elicited during an antitumor response. These differences include the utilization of alternative forms of signal transduction and alternative mechanisms of cytotoxicity.     

Alpha-glycosylceramides enhance the antitumor cytotoxicity of hepatic lymphocytes obtained from cancer patients by activating CD3-CD56+ NK cells in vitro

Alpha-glycosylceramides, such as alpha-galactosylceramide and alpha-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, V alpha 14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by alpha-glycosylceramides. We recently reported that V alpha 24 TCR+ NKT cells, the human homologues of murine V alpha 14 TCR+NK1.1+ cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether alpha-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of alpha-glycosylceramides in cancer immunotherapy in humans. Here, we show that alpha-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3-CD56+ NK cells. Even though V alpha 24 TCR+NKT cells proliferated remarkably in response to alpha-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of alpha-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3-CD56+ NK cells in the liver.     

Triggering of murine NK cells by CD40 and CD86 (B7-2)

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.