Cellular tumorigenicity in nude mice. Role of susceptibility to natural killer cells

The athymic nude mouse is a useful animal model for assaying the neoplastic growth potential in vivo of animal cells transformed in vitro. Despite the demonstrated absence of thymus-dependent immunological functions, however, the nude mouse has now been shown to reject transplants of certain highly malignant heterologous tumors. In addition, a few transformed mammalian cell lines that exhibit all or most of the cellular phenotypes usually associated with malignancy fail to grow as tumors when injected into nude mice. In a continuing study to identify the in vitro phenotypes associated with tumor-forming ability in vivo, we investigated the role of cellular susceptibility to the naturally occurring, thymus-independent lymphocytes (natural killer or NK cells) in determining tumor induction by animal cells in nude mice. A representative collection of animal cells (ranging from normal human diploid cell strains to highly tumorigenic clonal cell lines, either transformed in vitro or derived from experimental tumors) was tested to see if the ability of cells to form tumors is consistently correlated with their susceptibility to NK cell-mediated lysis measured in vitro with splenic leukocytes from nude mice. If the physiological role of the NK cells in vivo were to recognize, and possibly to destroy, incipient tumor cells in situ, a direct association between cellular tumorigenicity and susceptibility to NK activity, might be expected. If, on the other hand, the formation of growing tumors by animal cells in nude mice depended on their ability to escape the cytolytic activity of NK cells, cellular tumorigenicity would be associated with cellular resistance to NK cells. Results obtained in this study failed to confirm either of these associations. Thus, cellular suscepbibility to NK cells, at least as determined by direct cytotoxicity assay in vitro, is not a useful predictive indicator of cellular tumorigenicity in nude mice.     

Immunotherapy with ligands of natural killer T cells

Natural killer T (NKT) cells are innate lymphocytes that share receptor structures and functions with conventional T cells and natural killer cells. NKT cells are specific for glycolipid antigens bound by the major histocompatibility complex class I-like protein CD1d. One striking property of NKT cells is their capacity to rapidly produce large amounts of cytokines in response to T-cell receptor engagement, suggesting that activated NKT cells can modulate adaptive immune responses. Recent pre-clinical studies have revealed significant efficacy of NKT-cell ligands such as the glycolipid alpha-galactosylceramide for treatment of metastatic cancers and infections, and for prevention of autoimmune diseases. These findings suggest that appropriate stimulation of NKT cells could be exploited for prevention or treatment of human diseases.     

An absence of T cells in murine bone marrow allografts leads to an increased susceptibility to rejection by natural killer cells and T cells

The mechanisms behind the increased incidence of marrow graft failure in recipients that receive allogeneic marrow depleted of T cells were studied. Recipient mice were lethally irradiated and challenged with bone marrow cells (BMC) from C.B-17 +/+ (+/+) donors. Radioisotope 125IUdR incorporation was assessed 5 to 7 days after transfer to determine the extent of engraftment. Some groups received BMC in which the T cells were removed by treatment with antibody and C. In addition, some groups received BMC from T cell-deficient C.B-17 scid/scid (SCID) mice to determine the postulated need for donor T cells in hematopoiesis and engraftment. In a model system that distinguishes between possible host NK cell and radioresistant T cell-mediated rejection of marrow allografts, it was determined that the absence of donor T cells in a marrow graft does not affect engraftment in syngeneic recipients. However, both host NK cell and radioresistant T cell rejection was markedly enhanced when SCID BMC or BMC from C.B-17 +/+ donors that had T cells removed by antibody and complement were infused into irradiated allogeneic recipients. Furthermore, the addition of alloreactive thymocytes as a source of T cells could abrogate this increased susceptibility of the BMC to host rejection mechanisms. As determined by histology and 59Fe uptake, the addition of thymocytes resulted in enhanced erythropoiesis. These results suggest that the increased incidence of marrow graft failure when BMC depleted of T cells are used is a result of active rejection by host effector cells and that the adverse effect of marrow T cell depletion can be reversed by the addition of thymocytes.     

Scratching of their skin by NC/Nga mice leads to development of dermatitis

Effects of scratching behavior on dermatitis, transepidermal water loss (TEWL) and serum IgE concentrations were examined in NC/Nga (NC) mice with toenails (WIT) and without toenails (WOT). The first study was a preventive treatment done to cut off hind toenails before dermatitis induction and the second study was a therapeutic treatment by cutting off hind toenails of NC mice with severe dermatitis. In the preventive study, scratching behavior significantly increased in both WIT and WOT after dermatitis induction. Skin severity score, TEWL, number of mast cells and serum IgE concentration statistically increased in WIT but not in WOT after dermatitis induction. Histological changes coincided with the skin severity score in WIT, while no changes were observed in WOT. In the therapeutic study, skin severity score in WOT but not in WIT statistically decreased after cutting off the hind toenails. TEWL and numbers of mast cells in WOT were statistically lower compared with findings in WIT. Thus scratching up the skin with toenails seemed to be the most important factor leading to dermatitis in NC mice.     

Generation of cloned mice by direct nuclear transfer from natural killer T cells

Cloning mammals by nuclear transfer (NT) remains inefficient. One fundamental question is whether clones have really been derived from differentiated cells rather than from rare stem cells present in donor-cell samples. To date, cells, such as mature lymphocytes, with genetic differentiation markers have been cloned to generate mice only via a two-step NT involving embryonic stem (ES) cell generation and tetraploid complementation [1, 2 and 3]. Here, we show that the genome of a unique T-cell population, natural killer T (NKT) cells, can be fully reprogrammed by a single-step NT. The pups and their placentas possessed the rearranged TCR loci specific for NKT cells. The NKT-cell-cloned embryos had a high developmental potential in vitro: Most (71%) developed to the morula/blastocyst stage, in marked contrast to embryos from peripheral blood T cells (12%; p < 1 x 10(-25)). Furthermore, ES cell lines were efficiently established from these NKT-cell blastocysts. These findings clearly indicate a high level of plasticity in the NKT-cell genome. Thus, differentiation of the genome is not always a barrier to NT cloning for either reproductive or therapeutic purposes, so we can now postulate that at least some mammals cloned to date have indeed been derived from differentiated donor cells.     

Interferon-gamma upregulates the susceptibility of human neuroblastoma cells to interleukin-2-activated natural killer cells

The influence of interferon-gamma on the susceptibility of neuroblastoma cells in cell-mediated killing was investigated. Neuroblastoma cells were only weakly susceptible targets for peripheral mononuclear cells. However, enrichment of natural killer (NK) cells or activation of NK cells with interleukin-2 resulted in a considerable increase of neuroblastoma cell lysis. Pretreatment of neuroblastoma targets with interferon-gamma additionally increased the susceptibility to enriched NK cells as well as to interleukin-2-activated NK cells. The conjugate formation between enriched NK cells and the neuroblastoma targets was not affected by the pretreatment of the targets with interferon-gamma. Concomitantly, treatment of the neuroblastoma targets with interferon-gamma resulted in a strong induction of otherwise poorly expressed major histocompatibility complex (MHC) class I antigen expression. These results suggest that the increased expression of MHC class I antigens on target cells is not always correlated with decreased sensitivity for NK cells but can also be followed by an increased susceptibility for NK cells.     

N-acetylcysteine reduces susceptibility of transformed and embryonic cells to lytic activity of natural killer cells

Changes in the sensitivity of several transformed and embryonic cells to the lytic activity of natural killers (NK) due to N-acetylcysteine (NAC) has been studied. We found that epidermoid carcinoma A431 cells and murine hepatoma MH22a cells, as well as the transformed mouse fibroblasts 3T3-SV40 treated with 10 mM NAC that we investigated previously normalized their phenotype to the various extent. Like normal cells these cells exposed to NAC are not recognized and destroyed by NK. Murine embryonic fibroblasts (MEFs) similar to transformed cells were destroyed by NK. MEFs treated with 10 mM NAC lost their susceptibility to NK, as did transformed cells. The loss of cell sensitivity to NK cytolytic activity was accompanied by the reorganization of the actin cytoskeleton and generation of well-pronounced stress-fibers.     

Natural cytotoxic T cells (NCTC) that differ from natural killer (NK) and natural cytotoxic (NC) cells are present in Peyer's patches of mice

We have examined noninduced cytotoxicity of mouse gut associated and peripheral lymphoid tissues for a wide variety of syngeneic as well as allogeneic cell lines and lymphoblasts. Lymphoid cells from Peyer's patches were found to lyse these targets in a 3-hr chromium release assay whereas lymphoid cells from intestinal mucosa, mesenteric or peripheral lymph nodes, spleen, and thymus did not. The variety of targets toward which Peyer's patch cells were cytotoxic established the latter as nonspecific and H-2 unrestricted. The cell responsible for the lytic event was identified as possessing Thy 1.2 and Ia surface antigens. This naturally cytotoxic T cell (NCTC) was found to be adherent to nylon-wool but not to plastic plates. Although both natural killer cell (NK) and non-NK targets served as targets for the NCTC, the latter were further differentiable from NK cells by lack of asialo GM1 surface marker, which is present on NK cells. In addition, NCTC remained fully functional in mice given either of the drugs cyclophosphamide or cortisone. Each of these drugs, in the doses used, markedly reduced poly(I:C)-induced NK activity. Thus, NCTC differs from NK on the basis of the spectrum of targets against which it is functional, phenotypic surface markers, insusceptibility to stimulation with poly(I:C), and insensitivity to diminution by the immunosuppressive drugs cyclophosphamide and hydrocortisone. Since NCTC is a Thy 1.2 antigen-bearing cell and is detectable in a 3-hr cytotoxic assay, it also differs from the natural cytotoxic (NC) cell. NC lacks the Thy 1.2 marker and becomes detectable only in an 18-hr cytotoxic assay. Thus, NCTC is neither an NK nor an NC cell. We have discussed the possibility that the three naturally occurring cells may be related by being dedifferentiated descendants of an antigen-specific cytotoxic T lymphocyte (CTL). Alternatively, since NCTC is confined to an anatomical site prone to ample antigenic exposure and is still identifiable as a T cell, it may be in linear transition from the CTL to the NK or NC stages.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

The unique role of natural killer T cells in the response to microorganisms

Natural killer T (NKT) cells combine features of the innate and adaptive immune systems. Recently, it has become evident that these T cells have crucial roles in the response to infectious agents. The antigen receptor expressed by NKT cells directly recognizes unusual glycolipids that are part of the membrane of certain Gram-negative bacteria and spirochetes. Moreover, even in the absence of microbial glycolipid antigens, these T cells respond to innate cytokines produced by dendritic cells that have been activated by microbes. This indirect sensing of infection, by responding to cytokines from activated dendritic cells, allows NKT cells to react to a broad range of infectious agents.     

Enhancement of susceptibility of HSV-1-infected cells to natural killer lysis by interferon

The effect of interferon (IFN) on the natural killer (NK) activity of human PBL against HSV-1-infected HeLa cells was studied. Human PBL from several individuals did not consistently show a preferential lysis of HSV-1-, vaccinia-, or adenovirus type 5-infected cells with respect to uninfected HeLa cells. Treatment with IFN of effector PBL increased their lytic activity but did not alter the degree of preference on the lysis of the target cells shown by untreated PBL. Pretreatment with IFN of HSV-1-infected HeLa cells increased their susceptibility to lysis 5- to 10-fold. In contrast, identical pretreatment of the uninfected, adenovirus type 5- or vaccinia virus-infected HeLa cells before the assay decreased their susceptibility to NK lysis. This effect was not likely to be due to a block of the viral replication because other inhibitors like mitomycin C did not have the same effect. All target cells induced IFN synthesis in effector PBL cells. A similar level of IFN was induced by HSV-1-infected or uninfected HeLa cells. Pretreatment with IFN of HSV-1-infected, but not uninfected, HeLa cells induced 5 to 10 times more IFN by PBL, in good correlation with the increase in lytic activity. PBL treated with IFN, however, in conditions to give maximal stimulation of NK activity, presented the same preferential lysis of HSV-1-infected HeLa cells and synthesized similar levels of IFN as untreated PBL. In addition, HSV-1-infected HeLa cells were killed through different target structures than uninfected cells. Taken together, our results indicate an effect of IFN at the level of the NK target structures in HSV-1-infected HeLa cells by increasing either their number or, more likely, their affinity for NK cells independent of the effect of IFN in the effector cells or as an antiviral agent.     

Low numbers and altered phenotype of invariant natural killer T cells in recurrent varicella zoster virus infection

Invariant natural killer T (iNKT) cells play an important role in the immune response against various infectious agents. In this study we investigated their role in human defense against the varicella zoster virus. We observed decreased numbers of iNKT cells in patients who failed to control latent varicella zoster virus infection, e.g. underwent several reactivations of the virus. The residual population of iNKT cells expressed significantly higher levels of inhibitory receptor CD158a that was further up-regulated in the course of acute viral infection. Both of these abnormalities might contribute to impaired control of varicella zoster virus in human.     

Altered production and renewal of natural killer cells in B-lymphocyte-deficient CBA/N mice

The present study was designed to measure by quantitative and kinetic methods the production and renewal of natural killer (NK) cells in congenitally B-lymphocyte-deficient (CBA/N) mice. The total NK activity (percent specific lysis corrected for changes in whole organ cellularity) of the bone marrow and spleen of immunologically normal (CBA/CaJ) and CBA/N mice was assayed prior to and immediately after 48 h treatment (2 X/day, i.p.) with the cell cycle poison hydroxyurea (HU) and at various intervals throughout the subsequent post-HU recovery period. The total NK activity (TNKA) of untreated CBA/N bone marrow was 154% of that of CBA/CaJ bone marrow while the TNKA of CBA/N spleen was not significantly different (112%) from that of CBA/CaJ spleen. At the conclusion of 48 h HU, bone marrow TNKA of CBA/N and CBA/CaJ mice fell to 60 and 49%, respectively, of their saline-injected (2 X/day, i.p.) control levels, while spleen TNKA fell to 42 and 61%, respectively, of their saline-injected control levels. In the bone marrow, NK cell depletion in response to HU was more rapid in CBA/N mice (day 0.5 after HU) than in CBA/CaJ mice (day 2 after HU). TNKA of the spleen also decreased more rapidly in CBA/N mice (day 2 after HU) than in CBA/CaJ mice (day 3 after HU). The data indicate an enhanced production and turnover of NK cells in CBA/N mice relative to CBA/CaJ mice. Moreover, increased production and renewal of NK cells in CBA/N mice together with virtually unchanged levels of NK activity (112% of CBA/CaJ mice) in CBA/N mouse spleens indicate that mature lytic NK cells in CBA/N spleen but not bone marrow have a significantly shorter post-mitotic life span than do NK cells in the spleens of immunologically normal (CBA/CaJ) mice.     

The ability of konjac-glucomannan to suppress spontaneously occurring dermatitis in NC/Nga mice depends upon the particle size

Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions, which the higher viscous GM is proposed to have the stronger potencies. Application of highly viscous GM to various food industries is limited for the sake of long elapsed time required to solubilize the GM. Thus, the exploration into any physiological function of low-viscous GM remained undone for a long time. Male 4-week-old NC/Nga mice, a model of atopic dermatitis (AD), were fed for 8 weeks on diets containing 5% each of a high-viscous GM powder, ordinary konjac or highly purified GM, and a low-viscous GM powder, pulverized GM or re-granulated fine GM. Striking suppression to the aggravation of dermatitis and the increases in scratching behaviors and IgE levels was significantly observed only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. We concluded that the low-viscous GM possesses a novel immunoregulatory function and the intensity of the function depends upon the particle size rather than the viscosity.     

Expansion of unconventional T cells with natural killer markers in malaria patients

Immunological states during human malarial infection were examined. In parallel with parasitemia and anemia, granulocytosis was induced in the blood of patients, especially those infected with Plasmodium (P.) falciparum. At that time, the level of lymphocytes remained unchanged or slightly increased in the blood. However, the distribution of lymphocyte subsets was modulated, showing that the proportion of CD56(+)T cells, CD57(+)T cells, and gammadeltaT cells (i.e. all unconventional T cells) had increased in patients infected with P. falciparum or P. vivax. This phenomenon occurred at the early phase of infection and disappeared in the course of recovery. The data from patients with multiple attacks of P. vivax infection showed that there was no augmentation of these responses. In adult cases, the increase in the proportion of unconventional T cells seemed to closely parallel disease severity. However, all these responses were weak in children, even those infected with P. falciparum. In conjunction with accumulating evidence from mouse malaria experiments, the present results suggest that the immunological state induced by malarial infection might mainly be an event of unconventional T cells and that the immunological memory might not be long-lasting, possibly due to the properties of unconventional T cells.     

The regulatory role of natural killer T cells in the airways

Asthma, an inflammatory disorder of the airways, has been considered a disease mediated by allergen-specific Th2 cells and eosinophils, and controlled by allergen-specific regulatory T cells. This paradigm can explain many but features of asthma, but Th2 targeted therapies in patients with asthma have not been as successful as might be predicted by this paradigm. These observations have suggested that other cell types, such as Natural Killer T cells, may play an important role in asthma. In this review, we discuss the data that support the notion that NKT cells critically regulate the development of asthma.     

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1×10⁻⁷ M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1×10⁻⁷ M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear³H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of Il-2 through its ability to enhance the expression of intermediate affinity Il-2R on these cells.     

α‐galactosylceramide generates lung regulatory T cells through the activated natural killer T cells in mice

Our previous study showed that intraperitoneal injection of α‐galactosylceramide (α‐GalCer) has the ability to activate lung iNKT cells, but α‐GalCer‐activated iNKT cells do not result in airway inflammation in wild‐type (WT) mice. Many studies showed that iNKT cells had the capacity to induce Treg cells, which gave rise to peripheral tolerance. Therefore, we examined the influence of intraperitoneal administration of α‐GalCer on the expansion and suppressive activity of lung Treg cells using iNKT cell‐knockout mice and co‐culture experiments in vitro. We also compared airway inflammation and airway hyperresponsiveness (AHR) after α‐GalCer administration in specific anti‐CD25 mAb‐treated mice. Our data showed that intraperitoneal injection of α‐GalCer could promote the expansion of lung Treg cells in WT mice, but not in iNKT cell‐knockout mice. However, α‐GalCer administration could not boost suppressive activity of Treg cells in WT mice and iNKT cell‐knockout mice. Interestingly, functional inactivation of Treg cells could induce airway inflammation and AHR in WT mice treated with α‐GalCer. Furthermore, α‐GalCer administration could enhance iNKT cells to secrete IL‐2, and neutralization of IL‐2 reduced the expansion of Treg cells in vivo and in vitro. Thus, intraperitoneal administration of α‐GalCer can induce the generation of lung Treg cells in mice through the release of IL‐2 by the activated iNKT cells.