Natural Cell-mediated Cytotoxicity Research Articles

Interferon and interleukin-2 induced spontaneous cell-mediated cytotoxicity: a preliminary evaluation.

The contribution of natural cell-mediated cytotoxicity and its modulation with biological response modifiers in antitumour immunity was evaluated in patients with precancers and cancers of the oral cavity. The results were compared to those in normal controls. Both groups of patients showed highly significant depression of the natural killer cell activity. This depression was stage-related, suggesting an influence of tumour or tumour products on this activity. Depression in augmentation of this cytotoxicity was evident in both patient groups with cancers and precancers, suggesting a defect in the response of killer cells to biological response modifiers for reasons other than the tumour per se.

Augmentation of Natural Cell-Mediated Cytotoxic Activity by Supernatant fromIn VitroMitogen-Stimulated,In VivoHormone-Treated Lymphocytes in Immature Male (K Strain) Chickens

Newly hatched White Leghorn male chicks were used in this study. Triiodothyronine (T3; 0.1 or 1 ppm) and Thyrotropin Releasing Hormone (TRH; 1 or 5 ppm) were added to the feed for an 8-week period starting at hatch. A fifth group received the unsupplemented diet and served as controls. Peripheral blood lymphocytes from each treatment group were cultured in vitro with or without different mitogens (PHA, Con-A, or LPS), and the culture supernatants were tested for the presence of lymphokines (LK). The natural cell-mediated cytotoxicity (NCMC) assay was carried out with or without supernatant using the standard chromium (Cr51) release assay. Control untreated chicks were used as donors for effector cells and the P815 mouse mastocytoma was used as a target. Supernatant from in vivo 1 ppm T3- or 5 ppm TRH-treated lymphocytes significantly suppressed NCMC (or cells mediating NCMC, e.g., NK cells). However, supernatant from 1 ppm T3-treated, PHA-stimulated lymphocytes significantly enhanced NK cells cytotoxicity, while supernatant from 5 ppm TRH-treated lymphocytes with PHA stimulation tended to suppress cytotoxicity. These results provide evidence supporting a regulatory role of the hypothalamo-pituitary thyroid axis on lymphokine (LK) production. The results also suggest that these hormones act on different subpopulation of lymphocytes, and therefore, the mediators released by them.

Chicken growth hormone, triiodothyronine and thyrotropin releasing hormone modulation of the levels of chicken natural cell-mediated cytotoxicity

Newly hatched White Leghorn male chicks received dietary supplements of either Triiodothyronine (T 3) or Thyrotropin Releasing Hormone (TRH) until 8 weeks of age. Chicken growth hormone (cGH) (10 μg/kg BW) was injected into different chicks twice daily for 1 week starting at 7 weeks of age. Separate groups received both T 3 and cGH. Natural cell-mediated cytotoxic (NCMC) activity against different target cells was tested. It was found that cytotoxic activity in cells involved in NCMC against P815 mouse mastocytoma was stimulated by cGH alone or in combination with T 3 (1 ppm). These findings indicate that cGH and T 3 stimulate NCMC activity; and that the cells responsible for this activity may be Natural Killer (NK) cells.

Ultraviolet light-induced increase in tumor cell susceptibility to TNF-dependent and TNF-independent natural cell-mediated cytotoxicity

Previously we demonstrated that two consecutive in vitro irradiations of MCA 102 cells with high doses of UVC light (610 and 457 J/m 2) resulted in a selection of a permanent line MCA102UV that manifested high sensitivity to natural cell-mediated cytotoxicity (NCMC). In the present study analysis of the effector cells involved in lysis of these tumor cells was performed by comparing the cytotoxicity of normal spleen cells which mediated both NK and NC cell activity with (a) normal spleen cells in which NC activity was neutralized by anti-TNF Abs (NK +, NC −), (b) NK-depleted or NK-deficient spleen cells (NK −, NC +), and (c) NK-deficient or -depleted spleen cells with NC activity neutralized by anti-TNF Abs (NK −, NC −). Results of these studies indicate that lysis of the original MCA 102 tumor cells was relatively low and was mediated by NC cells. UV irradiation significantly increased MCA 102 tumor cell sensitivity to lysis by both NK and NC cells. Analysis of the mechanisms involved in UV-induced NK sensitivity revealed that UV irradiation increased tumor cell susceptibility to lytic NK-derived granules. NC sensitivity of MCA102UV tumor cells was associated with their increase in sensitivity to TNF and selection of MCA102UV cells for resistance to rTNF resulted in a decrease in their susceptibility to NC cells. To determine how fast UV-induced sensitivity to NCMC and rTNF can be established, 51Cr-labeled MCA 102 cells were irradiated in vitro with 38–304 J/m 2 of UVC light and their sensitivity to lysis by spleen cells and rTNF was tested immediately in an 18-hr cytotoxicity assay. UV treatment with the same doses was repeated 12 days later. The data obtained showed that tumor cell sensitivity to NCMC and TNF appeared shortly after UV irradiation, was stable, and was further substantially augmented by the second round of UV treatment. Thus, in vitro UV irradiation of tumor cells could be an effective modulator of tumor cell sensitivity to TNF-dependent and TNF-independent cell-mediated cytotoxicity.

Immunomodulation by 9-amino-1,2,3,4-tetrahydroacridine (THA): 1. Down-regulation of natural cell-mediated cytotoxicity in vitro

THA (Tacrine), a drug used in the experimental therapy of dementia of Alzheimer's disease type, and whose biochemical site of action is believed to be the neural cholinesterase, is shown, for the first time, to be an immunosuppressant in vitro on normal human peripheral blood lymphocytes in microgram quantities. THA down-regulates non-MHC restricted natural killer (NK) cell activity without affecting the general viability of cells. This down-regulation can be demonstrated at all effector and target (K562) concentrations, in purified resting NK cells as well as in lymphokine (interleukin 2) activated killer cells in 3- or 16-h NK assays and in all the blood samples tested. Kinetic analysis shows that the Vmax (maximal cytotoxic potential) and Km of NK cell-mediated cytolysis are also attenuated. Single cell assays using agarose matrix reveal that THA moderately interferes with tumor target binding/recognition events and strongly abrogates the delivery of lethal hit, thus lowering the frequency of active killer cells among THA-treated lymphocytes. THA down-regulates NK cells upon direct interaction and does not require the help of non-NK cells. The THA sensitive site(s) on NK cells does not appear to be perturbed significantly either by their proliferative status or by membrane modulations that may be normally induced by interleukin 2. The in vitro immunomodulatory pharmacological properties of THA reveal that the biological site of action of THA extends to non-neural cells also. Such non-neural models may be helpful in exploring the pathophysiological neuroimmunomodulatory properties of THA at cellular and molecular levels.

Human anti-murine immunoglobulin responses and immune functions in cancer patients receiving murine monoclonal antibody therapy

In our institution, over 200 patients with gastro-intestinal tract carcinomas have been treated with monoclonal antibodies (MAbs) including CO 17-1A. In one clinical trial, MAbs were administered in combination with gamma interferon. Natural killer cell cytotoxicity (NK) and antibody-dependent cell-mediated cytotoxicity (ADCC) were studied in patients before treatment. Very low NK and ADCC activities were measured in metastatic cancer patients. NK cell lysis was enhanced during gamma-interferon treatment, associated with a modification of the Fc receptor expression, but no changes in the ADCC reactivities of leukocytes were noticed. Monoclonal antibodies were circulating for one to four weeks after a single dose infusion, independent of the patients' immune responses toward the administered MAb. Sixty-three percent of the patients mounted an anti-mouse immunoglobulin response. Anti-idiotypic antibodies were detected in 70% of the responding patients. Variations in the anti-mouse Ig responses were dependent on the therapeutic protocol. The immune responses were composed of IgM, IgA, and IgG (mainly IgG1, often associated with IgG2 and/or IgG3). In patients receiving MAbs together with gamma-interferon, development of the anti-mouse Ig responses were delayed with an increase in the anti-isotypic component and a decrease in the anti-idiotypic component as compared to patients treated with MAb alone. No correlation could be established with clinical results.

Calcium-dependent natural killer and calcium-independent natural cytotoxic activities in an IL-2-dependent killer cell line.

Using a cloned murine cell line, NKB61A2, that concomitantly exhibits both NK and natural cytotoxic (NC) activities, we investigated the biochemical mechanisms involved in natural cell mediated cytotoxicity against NK-sensitive YAC-1 tumor cells and against the NC-sensitive WEHI-164 tumor cells. Recent reports have suggested that target cell lysis by cytotoxic lymphocytes occurs by either a calcium dependent and/or a calcium-independent mechanism(s). To determine the role of calcium in NK and NC activities of the NKB61A2 cell line, we evaluated the effect of: 1) extracellular Ca2+ depletion by the divalent cation chelator, EGTA, 2) Ca2+ influx blockade by the Ca2+ channel blocker verapamil, and 3) blocking of intracellular Ca2+ mobilization by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). We found that EGTA, verapamil, and TMB-8 were all capable of inhibiting NK activity, but they had little effect on NC activity of the NKB61A2 cells. Using 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide which are inhibitors of protein kinase C and calmodulin respectively, we determined that protein kinase C and calmodulin do play a role in the NK activity of NKB61A2 cells. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalanesulfonamide, similar to Verapamil and TMB-8, had no effect on NC activity. Thus, the data indicate that the NK activity of NKB61A2 cells is calcium dependent whereas NC activity is not. These results may explain the disparate reports seen in the literature of calcium-dependent and -independent lysis of tumor cells.

Increased sensitivity to MHC-nonrestricted lysis of BL6 melanoma cells by transfection with class I H-2Kb gene.

Abstract An H-2Kb- negative clone of BL6 melanoma (BL6-8) was transfected with neor, H-2Kb, or H-2IAk genes. In an 18-h cytotoxicity assay clones with high levels of H-2Kb Ag expression were found more sensitive to lysis by spleen cells of syngenic and allogeneic mice than H-2Kb low clones. NK cells were involved in the lysis of H-2Kb+ BL6 melanoma clones, with spleen cell cytotoxicity of mice increased after poly I:C stimulation or decreased after pretreatment with anti-asialo GM1 serum or NK1.1 mAb. Anti-TNF Ab were also able to reduce the cytotoxicity of normal spleen cells and completely abolished the cytotoxicity of the NK-depleted spleen cells suggesting involvement of NC cells in lysis of H-2Kb+ BL6 melanoma clones. Increase in sensitivity of H-2Kb+ BL6 cells to natural cell-mediated cytotoxicity was associated with the appearance of NK recognizable determinants as assessed by the cold target inhibition assay. All BL6 clones, irrespective of sensitivity to natural cell-mediated cytotoxicity, showed high sensitivity to lysis by LGL-derived granules. In contrast, all H-2Kb low BL6 clones were resistant and all H-2Kb highly positive clones were sensitive to lysis by TNF-alpha. When an H-2Kb highly positive clone was selected in vitro for resistance to TNF, it concomitantly showed increased resistance to cytotoxicity by spleen cells, confirming the importance of TNF in spleen cell cytotoxicity against H-2Kb+ melanoma cells. Taken together, the data indicate that class I H-2Kb but not class II H-2IAk gene product could increase the sensitivity of BL6 cells to lysis by NK and natural cytotoxic cells as well as TNF. We hypothesize that these effects could be due to pleiotropic effects of H-2Kb gene products on various biologic properties of BL6 melanoma cells some of which may be more directly involved in regulation of tumor cell sensitivity to lysis by NK and/or natural cytotoxic cells.

Immunotoxicity of Bis(tri- n-butyltin)oxide in the rat: Effects on thymus-dependent immunity and on nonspecific resistance following long-term exposure in young versus aged rats

To investigate whether immune function suppression observed in an earlier study after short-term bis(tri- n-butyltin)oxide (TBTO) exposure also occurred after long-term treatment, function studies for specific and nonspecific resistance were performed after exposure of weaned male rats to diets containing 0, 0.5, 5, or 50 mg TBTO/kg for 4–6 and 15–17 months. Treatment for 4.5 months had no effect on body weight but reduced thymus weight at 50 mg/kg. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin and tuberculin were not depressed, in contrast to the results of the short-term study. The resistance to the nematode Trichinella spiralis was dose-relatedly suppressed at the 5 and 50 mg/kg levels, in both experiments (5.5 and 16.5 months exposure), as shown by increased counts of muscle larvae and depressed serum IgE titers. Also the inflammatory reaction around cysts in parasitized musculature was reduced. No significant reduction was found in IgM and IgG titers to T. spiralis, ovalbumin, and sheep red blood cells as determined by enzyme-linked immunosorbent assay. TBTO exposure at 50 mg/kg for 4.5 months significantly reduced thymus weight, but the response of thymocytes to T-cell mitogens was unaltered. TBTO treatment for 4.5 or 16 months did not influence the response of spleen cells to T- and B-cell mitogens and neither influenced spleen weight. A dose-related shift was observed in T- and B-cell numbers in mesenteric lymph nodes as shown by flow cytometry using monoclonal antibodies: treatment for 6 and 18 months reduced the relative count of T-lymphocytes and consequently increased the percentage of B-lymphocytes. As a result, the T:B ratio was reduced in the 5 and 50 mg/kg groups. Concerning the nonspecific resistance, TBTO exposure for 5 and 17 months reduced macrophage function at 50 mg/kg as shown by impaired splenic clearance of Listeria monocytogenes bacteria. Natural cell-mediated cytotoxicity of spleen and peritoneal cells was investigated in a 51Cr-release assay with YAC-lymphoma target cells. TBTO treatment significantly suppressed natural killer (NK) activity in spleen cells. Significant suppression was noted in all treatment groups following 16 months TBTO exposure; in contrast to treatment for 4.5 months. No significant alterations were observed in the spontaneous cytotoxicity of nonadherent and adherent peritoneal cells following 4.5 months treatment. Treatment of aged (i.e., 1-year-old) male rats for 5 months with the 50 mg/kg diet reduced thymus weight but had no effect on body or spleen weight. At this dose level the resistance to T. spiralis was suppressed as shown by a retarded expulsion of adult worms from the small intestine and increased counts of muscle larvae. Also the splenic clearance of L. monocytogenes bacteria was reduced at the 50 mg/kg dietary level. It is concluded that long-term TBTO exposure of young and aged rats suppressed host resistance to a bacterial ( L. monocytogenes) and especially to a parasitic ( T. spiralis) infection, although the effects in aged rats were less pronounced. These data demonstrate the sensitivity of these challenge models since alterations were detected at dose levels at which no other effects were found.

Bovine natural cell mediated cytotoxicity (NCMC): Activation by cytokines

Incubation of bovine peripheral blood mononuclear leukocytes (PBML) with the cytokines (CK) IL-2, α-IFN, γ-IFN or IL-4 resulted in significant increases in natural cell mediated cytotoxicity (NCMC) over endogenous levels, as determined in an 18 h 51Cr-release assay using the human K562 or mouse Yac-1 target cell lines. Endogenous cytotoxic activity of bovine natural effector cells (NEC) using K562 or Yac-1 target cells was minimal (killing <8%). After 18 h of incubation with the CK hu rIL-2, α-bov rIFN, γ-bov rIFN or hu rIL-4, NEC had significant increases in cytotoxic activity for both K562 and Yac-1 target cells. Significant increases in cytotoxic activity were not found after incubation of NEC with IL-1 or β-IFN. Specific killing varied with CK concentration in a dose dependent manner and was proportional to effector: target cell ratio. Activation of the bovine NEC by CK was rapid, occurring within 6–12 h of incubation with α-IFN or γ-IFN and within 12–18 h of incubation with IL-2. Incubation of bovine PBML with IL-2 and α- or γ-IFN or with α-IFN and γ-IFN showed that these CK do not act in a synergistic manner to increase NCMC in the bovine NEC.

Natural cell-mediated cytotoxicity against various target cells in patients with epidermodysplasia verruciformis

Natural cell-mediated cytotoxicity of peripheral blood mononuclear cells was studied in eight patients with epidermodysplasia verruciformis induced by human papillomaviruses specific for epidermodysplasia verruciformis and in five patients with epidermodysplasia verruciformis-induced exclusively by human papillomavirus type 3. Nine patients with various cutaneous warts and 25 age- and sex-matched healthy persons were control subjects. Natural cell-mediated cytotoxicity against both K-562 erythroleukemic and Sk-v cells was in the normal range in patients with epidermodysplasia verruciformis induced by epidermodysplasia verruciformis-specific human papillomaviruses and in patients with cutaneous warts. The lysis of both targets, however, was significantly decreased in patients with the form of epidermodysplasia verruciformis associated with human papillomavirus type 3. Experiments with normal keratinocytes and with keratinocytes isolated from a malignant lesion bearing human papillomavirus type 5 genomes showed that the latter were susceptible to lysis by the peripheral blood mononuclear cells of healthy persons and of patients with cutaneous warts. Lysis of keratinocytes in epidermodysplasia verruciformis, however, was strongly reduced in patients with epidermodysplasia verruciformis induced by specific human papillomaviruses. This reduction was not associated with a decrease in anti-K-562 natural cell-mediated cytotoxicity. Our results suggest that in patients with epidermodysplasia verruciformis induced by disease-specific human papillomaviruses, there is reduced natural cell-mediated cytotoxicity against epidermodysplasia verruciformis keratinocytes.

Role of Vincristine in Immunochemotherapy of Leukemia in Mouse or Human Models

Synergistic antitumor effects between Vincristine (VCR) and allograft responses have been found in mice bearing allogeneic retrovirus-induced leukemia. In this model VCR depressed weakly allograft reactivity if given before but not after antigen administration. In a parallel human tumor model in vitro using HTLV-1 induced MT-2 leukemia, additive but not synergistic immuno-chemotherapeutic effects were obtained with allogeneic mononuclear cells (MNC) combined with VCR at 0.1 but not at 1 micrograms/ml. In this case natural immunity (NI) rather than antigen-dependent immunity (ADI) was involved in the combined effects of VCR + MNC. In the in vitro model pretreatment of effector cells with 1 or 0.1 micrograms/ml of VCR depressed natural cell-mediated cytotoxicity (NCMC). However when the drug was added to the effector + target cells during the 4 h cytotoxicity assay, 1 but not 0.1 micrograms/ml of the drug was capable of depressing NCMC function. These results would provide valuable information for developing in vitro immuno-chemotherapy studies in human tumor systems, including those characterized by the presence of tumor-associated oncogenic retroviruses, capable of depressing both NI and ADI functions.

The influence of interferon alpha and gamma, singly or in combination on human natural cell mediated cytotoxicity

The influence of interferon alpha and gamma alone or in combination on the augmentation of human natural cytotoxicity was studied. Treatment of peripheral blood lymphocytes with IFN- alpha led to a rapid augmentation of NK activity, in contrast to IFN-gamma where target cell killing was observed only following 18 hrs exposure of lymphocytes to IFN-gamma. The results of the single cell assay paralleled those obtained using the Chromium release test, but neither interferon type caused an increase in the number of target binding lymphocytes. The combined effect of IFN-alpha and IFN-gamma in stimulating human natural cytotoxicity demonstrated individual lymphocyte responses to be variable. Exposure of lymphocytes to IFN-alpha and IFN-gamma for 18 hrs prior to assay for cytotoxicity usually decreased the level of cytotoxicity compared with control values, whereas other treatment regimes gave an additive and sometimes synergistic effect. Only treatment with IFN-alpha for 18 hrs and IFN-gamma for one hr produced a synergistic response in the majority of individuals tested. We conclude from this study that individual responses to IFN-alpha and IFN-gamma alone or in combination are variable and dependent upon timing of exposure of lymphocytes to individual interferon types, and possibly reflects the donor status at the time of sampling.

Autologous and allogenic natural cell mediated cytotoxicity at the single cell level: divergent effects of interferons and interleukin 2 on binding and lysis.

In order to evaluate their divergent effects on binding and lysis of the NCMC, rH IFN-alpha and rH IL-2 were used for the in vitro-preincubation of normal donors' PBL, which were then tested as effector cells against K562 and the long-term cultured melanoma cell line RIMA in the SCCA. Both lymphokines significantly augmented the cytolysis of K562 without relevant influence on the conjugate formation. However, against RIMA IFN-alpha additionally amplified the binding affinity. This in keeping with the other authors' opinion that IFNs have target-specific effects at the single cell level, with the principal activation of already conjugated pre-killer cells against all the target cell lines tested. We performed a therapy-follow-up of the i.p. administration of rH IFN-gamma to patients with ovarian carcinomas in vivo. With the SCCA we detected a significant correlation of the duration of therapy with the autologous cytotoxicity in the ascitic compartment. In one case the increase of this parameter was even exponential. However, the countercurrent trend of the conjugate formation delayed and reduced the activation of the autologous total killer activity. This relative stagnation resulted in the inadequate clinical response of two patients. Moreover, we observed a discrepancy in the development of the autologous and allogeneic SCCA-parameters suggesting strong effects of the peritumorally administered IFN directly on the effusion tumor cells.

Non-Hodgkin's lymphoma: natural cell-mediated cytotoxicity correlated with histological classification and prognosis.

Non-Hodgkin's lymphoma (NHL) patients classified according to the Rappaport classification system had depressed natural killer (NK) and NK cytotoxic factor activities, antibody-dependent cellular cytotoxicity, active killing potential as well as recycling capacity. The suppression of these activities was not related to the favorable or unfavorable prognosis of the patients. Paramaters of the NK cytotoxic status of the NHL patients did not correlate with the histology or prognosis of the disease.

Induction of lymphokine-activated killer-like cells by cancer chemotherapy.

Natural cell-mediated cytotoxicity against NK-resistant target tumor cells was found in the peripheral blood of tumor-bearing patients approximately 1 mo after combined chemotherapy. The recognition specificity of these effector cells was broad and had no restriction. From the experiments of negative selection with mAbs and complements, these newly developed killer cells after chemotherapy were thought to be LAK-like cells. Contribution of these LAK-like cells to the mechanism of action of anticancer drugs remains to be clarified.

Murine trophoblast resists cell-mediated lysis: II. Resistance to natural cell-mediated cytotoxicity

The susceptibility of murine trophoblast cells to natural cell-mediated cytotoxicity has been assessed. Primary short-term cultures of murine trophoblast cells isolated from 14-day placentas were found to be resistant to endogenous and interferon-activated natural killer (NK) cells and natural cytotoxic cells. That the relevant target structures are expressed on the surface of trophoblast cells and accessible to the effectors was demonstrated by their ability to inhibit the lysis of NK-sensitive target cells (YAC-1) in a dose-dependent manner. The lytic resistance of trophoblast cells was unaffected by neuraminidase treatment, inhibition of protein synthesis, or extending the assay time to 12 hr. Moreover, trophoblast cells were resistant to antibody-dependent cell-mediated cytotoxicity when coated with an alloantibody capable of mediating their lysis in the presence of heterologous complement. Neither the preincubation of effector cells in concentrated trophoblast culture supernatants nor the direct exposure of effectors to monolayers of trophoblast cells inhibited their NK lytic activity, indicating that the secretion of a suppressive factor or the direct inactivation of the NK cells was not responsible for the observed resistance to lysis. These observations, together with previous results showing the resistance of trophoblast to cytotoxic T cell-mediated lysis, reveal that murine trophoblast cells possess a resistance mechanism against several forms of cell-mediated lysis. This feature of trophoblast cells at the maternal-fetal interface is likely to play an important role in protecting the fetoplacental allograft from immune rejection.

Immune response to ultraviolet-induced tumors. II. Effector cells in tumor immunity.

Skin tumors induced in mice by chronic ultraviolet irradiation are highly antigenic and can induce a state of transplantation immunity in syngeneic hosts. In the present study, we compared the in vitro cytolytic activity of splenic lymphocytes from mice immunized with either a regressor or a progressor UV-tumor. The results of this comparison supported previous work implicating a role for tumor-specific cytolytic T lymphocytes in the rejection of regressor UV-tumors. The results also revealed that immunization with the progressor UV-tumor 2237 failed to elicit detectable levels of progressor tumor-specific CTL in animals capable of rejecting the immunizing tumor. Interestingly, following in vitro resensitization of both regressor and progressor immune spleen cells, we found a previously undetected lymphocyte population with anti-UV-tumor activity. Besides lysing UV-tumors in vitro, these lymphocytes also lysed a wide variety of additional tumor targets. This effector activity along with the analysis of cell surface markers indicated that these lymphocytes belong to that category of effector cells mediating natural-cell-mediated cytotoxicity (NCMC). As we had not detected cells with this activity in splenic lymphocyte preparations prior to in vitro resensitization, we examined lymphocytes from the local tumor environment during the course of progressor 2237 tumor rejection for either NCMC activity or tumor-specific CTL activity. This in situ analysis revealed lymphocytes exhibiting significant levels of cytolytic activity against several UV-tumors, thus implicating NK cells as effector cells in the rejection of progressor UV-tumors by immune animals. The mechanisms whereby NK cells with NCMC activity could be induced in immune animals are discussed in the context of class-II-restricted immune responses by helper/inducer T lymphocytes.

Immune response to ultraviolet-induced tumors. III. Analysis of cloned lymphocyte populations exhibiting antitumor activity.

Previously, we hypothesized that natural killer lymphocytes could function as effector cells in the rejection of UV-induced tumors in tumor-immune animals. Immunization with progressor UV-tumor 2237 induced lymphocytes exhibiting natural cell-mediated cytotoxicity but failed to elicit tumor-specific cytolytic T lymphocytes. In the present investigation, T lymphocyte cloning technology provided a means of isolating homogeneous lymphocyte populations exhibiting CTL and NK activities. Clones with both CTL and NK activity were isolated from regressor-1316-immune mice, but NK-like clones only were isolated from progressor-2237-immune mice. An evaluation of the in situ anti-UV-tumor action of a representative NK lymphocyte clone revealed that these cells could in fact prevent tumor outgrowth, supporting our hypothesis that these cells could function as effector cells in UV-tumor rejection responses in tumor-immune animals.

Effects of lentinan on cytotoxic functions of human lymphocytes.

The in vitro effects of lentinan on natural killer (NK), antibody-dependent cell-mediated cytotoxicity (ADCC), lectin-dependent cell-mediated cytotoxicity (LDCC) and mitogen-induced blast transformation were studied in patients with solid tumors and chronic lymphocytic leukemia (CLL). NK activity was measured against 51Cr-labelled K-562 targets, ADCC against antibody-coated chicken red cells. LDCC and natural cell-mediated cytotoxicity (NCMC) was assessed using 3H-thymidine prelabelled HEp-2 targets. Mitogen (PHA- and Con A-) induced blast transformation was measured by thymidine incorporation. Blastogenesis and LDCC was not influenced by lentinan. 1 microgram/ml lentinan increased NCMC of tumor-bearing subjects. The most prominent enhancement of NK and ADCC activity was seen in CLL patients, where a dose-related increase was seen (from 0.01 to 1 microgram/ml).