In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

lack of suppressor cell activity for natural killer cells in infant, aged and a low responder strain of mice

We have examined the reported role of suppressor cells in the regulation of NK activity in mice with naturally low NK activity (infant and aged (C57 X A)F1 hybrids (CAF1) and low responder strain AKR mice). Possible suppressor activity was assayed by mixing, at a 1 : 1 ratio, spleen cells from low activity mice with spleen effector cells from normally active 8 to 10 wk old CAF1 mice. The lytic activity of the mixture was compared with the activity of effector cells diluted with medium alone or diluted 1 : 1 with "non-suppressor" population which served as a control for nonspecific decreases in lysis. The control or "filler" cells employed were suspensions of adult CAF1 thymus, thymus from adult mice exposed to 2,000 R, and adult CAF1 spleen cells cultured for 24 hours, a procedure that depleted NK activity. In no case was the activity observed in the presumed suppressor-effector mixture significantly lower than that observed in the filler-effector cell mixtures. Thus, in infant (1 to 2 wk) and aged (12 to 18 mo) CAF1 mice and in 8 to 10 wk old AKR mice, we found no evidence for specific cell-mediated suppression of natural cytotoxicity.     

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF)

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.     

Sensitivity of simian virus 40-transformed C57BL/6 mouse embryo fibroblasts to lysis by murine natural killer cells

The susceptibility of mouse cells expressing full-length or truncated transforming protein (T antigen) of simian virus 40 (SV40) to lysis by murine natural killer (NK) cells was assessed. For these studies, C57BL/6 mouse embryo fibroblasts (B6/MEF) were transformed by transfection with SV40 DNA encoding the entire T antigen. The transformed cell lines were tested for susceptibility to lysis by nonimmune CBA splenocytes as a source of NK cells and to lysis by C57BL/6, SV40-specific cytolytic T cells (CTL). It was found that 13 of 15 clonally derived, SV40-transformed H-2b cell lines were susceptible to lysis by NK cells. However, there was some variation in their susceptibility to lysis by NK cells. There was no correlation between susceptibility to lysis by SV40-specific CTL and to lysis by NK cells. Cells transfected with a plasmid which encodes only the N-terminal half of the SV40 T antigen were consistently less susceptible to lysis by NK cells, suggesting that expression of only the N-terminus of the T antigen was insufficient for optimal susceptibility to lysis by NK cells. Primary mouse embryo fibroblasts transformed by human adenovirus type 5 E1 region DNA were also found to be susceptible to NK cell-mediated lysis. Lysis of SV40-transformed cells by nonimmune CBA splenocytes was mediated by NK cells because: lysis was augmented when the effector cells were treated with interferon before assay; and lysis was abrogated when the effector cells were obtained from mice that had been depleted of NK activity by treatment with antiserum against the asialo GM1 surface marker. These results indicate that primary mouse cells which are transformed by SV40 and which express the native T antigen are susceptible to lysis by mouse NK cells. Conversely, cells transformed by a plasmid encoding only the N-terminal half of the T antigen express reduced susceptibility to lysis by NK cells.     

Natural killer cells in rhesus monkeys: properties of effector cells which lyse Raji targets

Spontaneously occurring natural killer cell activity of rhesus monkey peripheral blood mononuclear cells was assayed against five human cell lines, three of which were Epstein-Barr virus (EBV) positive, including the human B cell line Raji. The lytic activity to Raji cells was high, significantly higher than to any other cell line tested. Raji cells are normally insensitive to spontaneous lysis by human NK cells, and the contrasting vigor of the rhesus monkey cytolytic activity to Raji prompted us to investigate the properties of this effector cell. We found the effector cell-mediating lysis of Raji to be nonadherent and phagocytic with lytic activity slightly enhanced in the E-rosette-forming cell (ERFC+) fraction and decreased in the ERFC- fraction. Further isolation of FcIgG receptor-positive and FcIgG receptor-negative subsets by rosetting resulted in significant enrichment of NK activity to Raji in the positive fraction and a loss of activity in the negative fraction. Depletion studies with various monoclonal antibodies (mAb's) confirmed that nearly all lytic activity was contained in the CD16+ (Leu 11b+) population, while subsets of effector cells expressed CD2 (9.6) and CD8 (OKT8). Depletion of CD4 (OKT4)-, HLADR (OKIa)-, or LFA1 (MAC-1)-positive populations failed to reduce NK activity. We compared the phenotypic properties of alloimmune effector cells exhibiting specificity for allogeneic donor targets with those exhibiting lysis of Raji targets. Results indicated that allospecific cytotoxic T lymphocytes expressed a CD16-, CD2+ phenotype, a pattern distinct from that of the effector cell population recognizing Raji targets. The presence of CD2 mAb's in the culture had no effect on NK lytic activity. In contrast, mAbs CD8 and Leu 11b were inhibitory. This would suggest a functional role for CD8 and FcIgG molecules in the lysis of Raji cells by rhesus effectors. In summary, these studies describe a distinct population of effector cells in the blood of rhesus monkeys which exhibit spontaneous lytic activity to Raji cells and exhibit the properties of NK cells.     

H-2 antigen expression and sensitivity of BL6 melanoma cells to natural killer cell cytotoxicity

The sensitivity of H-2b-high and H-2b-low variants of BL6 melanoma to the cytotoxic action of NK and lymphokine-activated killer cells was investigated. BL6 mouse melanoma cells lack detectable H-2Kb and had low levels of expression of H-2Db Ag. The BL6T2 variant cells, obtained after treatment of BL6 cells with mutagen N-methyl-N-nitro-N'-nitro-soguanidine, had relatively high levels of expression of class I H-2b Ag. Poly(I:C)-stimulated spleen cells of nude mice were highly cytotoxic for BL6T2, whereas H-2b-low BL6 cells were less sensitive to NK activity in an 18-h 51Cr-release assay. Similar results were obtained after 4-h incubation of radio-labeled tumor cells with IL-2-activated effector cells. In contrast, both lines were equally sensitive to lysis by purified granules derived from rat large granular lymphocytes (LGL) or by macrophages. By using various clones selected from BL6 or BL6T2 cells, it was found that BL6 or BL6T2 clones with low H-2b Ag expression were less sensitive to lysis by NK cells than H-2b-high clones. After IFN treatment of either BL6 or BL6T2, the target cells became more resistant to lysis by either NK cells or by purified LGL granules. IFN-treated BL6 cells had substantially increased expression of H-2b Ag and in this respect became similar to untreated BL6T2. However, IFN-treated BL6 cells were more resistant than BL6T2 cells to lysis by NK cells and LGL granules, suggesting that augmentation of H-2b Ag expression and NK resistance could be two independent IFN-induced effects. With a cold target inhibition assay, it was found that BL6T2 or its H-2 positive clones were highly competitive and inhibited the cytotoxic activity of NK and lymphokine-activated killer cells against radiolabeled YAC-1 and BL6T2, whereas BL6 cells or H-2-negative clones of BL6T2 and BL6 lines showed poor competitive ability. Thus, our data indicate that the NK resistance of H-2-low BL6 cells may be due to a paucity of NK recognizable determinants. N-Methyl-N-nitro-N'-nitroguanidine treatment of BL6 melanoma cells was associated with an increase in class I H-2b Ag expression and NK sensitivity, suggesting the involvement of class I MHC Ag in the sensitivity of tumor cells to NK cell-mediated cytotoxicity.     

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.     

Renal carcinoma cell lines inhibit natural killer activity via the CD94 receptor molecule

MHC class I molecules protect normal and transformed cells from lysis by natural killer (NK) cells through recognition of receptors expressed on leucocytes. Defects in NK cell activity and lymphokine activated killer (LAK) cell generation have been previously demonstrated in patients with renal cell carcinoma (RCC). However, to date, the importance of NK receptor/MHC class I interactions for immune evasion by RCC cells has not been described. In this study, human RCC cell lines (HTB46, HTB47, ACHN, CRL 1933 and HTB44) were found to be susceptible to lysis by both NK cells and interleukin-15 (IL-15)-derived LAK cells from normal donors in vitro. However, when NK cells were co-cultured with RCC cells their expression of the CD94 NK receptor molecule was significantly increased and their cytolytic activity against RCC targets was reduced. The cytolytic activity of NK cells was restored by the addition of IL-15, which further augmented the expression of CD94 on CD56+ NK cells. Disruption of NK receptor-MHC class I interactions by the addition of blocking antibodies to CD94 had no effect on the lysis of K562 or HTB47 targets by NK cells. However, the sensitivity of HTB46 cells to NK-mediated lysis was increased by blocking the CD94 receptor molecule, but only when the NK cells had not been previously co-cultured with RCC cells. This was independent of the presence of IL-15. These results show that RCC cells can inhibit NK activity via CD94 and suggest that disruption of interactions between receptor and ligand on RCC cells in vivo may augment the immune response against tumours by innate effector cells.     

HER-2/neu peptide specificity in the recognition of HLA-A2 by natural killer cells

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide–MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2⁺ or A2⁻). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells. Received: 16 March 1999 / Accepted: 3 June 1999     

Purification and target cell range of in vivo elicited blast natural killer cells

Blast natural killer (NK) cells were elicited in the spleens of mice by treatments with the interferon inducers lymphocytic choriomeningitis virus (LCMV) or polyinosinic-polycytidylic acid (poly I:C). The blast-NK cells, separated on the basis of size by centrifugal elutriation, were compared with blast cytotoxic T lymphocytes (CTL) generated during infection with LCMV. In vivo treatments with antibody to asialo GM1 (AGM1) blocked the appearance of blast-NK cells but not blast-CTL. Antibody and complement depletion experiments indicated that the blast-NK cells were AGM1+, NK 1.2+/-, Lyt-5+/-, Thy+/-, Qa-5/NK 1.1+, Lyt-2-, B23.1-, and J11d-. Blast-NK cells could be unequivocally distinguished from blast-CTL, because the blast-CTL were completely sensitive to treatments with anti-Lyt-2 and complement, whereas the blast-NK cells were completely resistant. The blast-NK cells were purified from populations of large-size cells by antibody and complement treatments that depleted the co-eluting monocyte/macrophages and polymorphonuclear leukocytes. The population resulting after separation from dead cells over Percoll gradients represented approximately 1% of the total spleen cells, contained greater than 60% large granular lymphocytes and mediated greater than 15% killing of YAC-1 target cells in a 4-hr 51Cr release assay at an effector to target cell ratio of 1:1. The purified blast-NK cells lysed a broad range of target cells at relatively low effector to target cell ratios. The order of sensitivity of the target cells was YAC-1 much greater than K562 approximately equal to L-929 much greater than P815, consistent with that reported for NK cell-mediated lysis. The ability of the blast-NK cells to mediate lysis of NK cells also was examined. The purified NK cells mediated significant levels of lysis against the NK-like cloned line, NK1B6B10, in a 51Cr release assay. Furthermore, the purified blast-NK cells mediated lysis of bound blast-NK cells in a single-cell agarose assay. These results indicate that highly purified blast-NK cells are exceptionally efficient at mediating lysis and suggest that NK cells may act to negatively regulate the proliferation of NK cells by lysing other NK cells.     

Monoclonal antibody 4H12 recognizes subsets of adherent-lymphokine activated killer cells and splenic natural killer cells from pregnant and neonatal mice

Using a new mAb, 4H12, that recognizes a plasma membrane-associated Ag on granulated metrial gland cells, we identified subsets of murine NK cells in spleen cell-derived adherent lymphokine activated killer cells and in the spleens of neonatal and pregnant mice. In spleen cell adherent-lymphokine-activated killer cultures, 4H12 Ag was detected on a small subset of cells after 7 days culture and expression increased with time to 70% of the cells after 21 days culture. 4H12+ cells were large (up to 70 microns) and granular. The Ag was also detected in the cytoplasmic granules of some, but not all 4H12 surface positive cells. Coexpression studies indicated 4H12+ cells were predominantly positive for the NK1.1 and ASGM1 Ag, negative for the MAC-1 and F4/80 Ag, and +/- for the LGL-1 and CD3 Ag. Subsets of 4H12+/-, LGL-1+/- exhibited morphologic characteristics restricted to specific phenotypic subsets. The 4H12-/LGL-1+ subset was shown to contain the smallest, least granular cells, whereas the 4H12+/LGL-1+/- subsets contained the largest and most granular cells. Although 4H12 expression was negligible in the spleens of normal adult mice, spleen cells of neonatal and pregnant mice contained subsets of NK1.1+ cells that coexpressed 4H12. The 4H12+/NK1.1+ and 4H12-/NK1.1+ subsets displayed differential levels of NK1.1 expression. 4H12+ NK cells were NK1.1 low to high, whereas 4H12- NK cells were NK1.1 high only. The functional significance of subsets of NK cells in IL-2 culture and in the spleens of neonatal and pregnant mice remains to be elucidated.     

Susceptibility to lysis by natural killer and natural cytotoxic cells is independent of the mitotic stage of the target cell cycle

Natural cell-mediated cytotoxicity (NCMC) against a number of target cells is mediated by at least two distinct effector populations, with natural killer (NK) and natural cytotoxic (NC) cells being the predominant in the murine system. The studies described in this report examine the role that the phase of the mitotic cycle of the target cell has on its susceptibility to lysis by NC and NK cells. We show that neither the kinectics nor the magnitude of NC cell lysis is altered when assayed using target cells which have been enriched for G1, S, or G2 + M stages of the cell cycle. Similarly, NK cell lysis by fresh or poly-IC augmented effector cells was not effected by target Cell Cycle.     

Missing self by heterogeneous natural killer cells

Some murine (YAC, P815 and SP20) and human (Molt4, Raji and HR7) tumour cell lines were (i) treated with IFN-γ for inducing enhanced expression of MHC class I antigen, or (ii) given a brief treatment with citrate buffer (pH 3.0), which resulted in denaturation of class I MHC antigens on these tumour cells. IFL-γ or acid treated tumour cells were used as unlabelled competing targets in cold target inhibition assays. The results indicated that the competing ability of acid-treated tumour cells remained unaltered, whereas IFN-γ treated tumour cells competed with significantly less efficiency. These results have been evaluated in light of the current view of NK cell development and the expression of inhibitory receptors for MHC class I molecules (IRMs), on NK cells. A modified view on NK cell heterogeneity based upon IRM expression has been proposed which reconciles several apparently discordant observations about the activity and role of NK cells. Two classes of NK cells have been proposed. Type I NK ceils have target recognition receptors which do not recognize autologous normal cells, lack IRMs, and may participate in first line of defence against transformed cells in vivo. Type II NK cells have target recognition receptors for autologous normal cells and express at least one self-reactive IRM in order to prevent auto-killing. Type II NK cells participate in killing those transformed cells which down-regulate their MHC class I expression in order to escape cytotoxic T-cell surveillance. It is also postulated that mechanism of inverse correlation of target cell MHC class I expression levels and their susceptibility to NK cells, involves interference model of missing self hypothesis for type I NK cells and inhibitory signal model of missing self hypothesis for type II NE cells. Finally, it is proposed that acid treatment of tumour cells enhances their lysis susceptibility by making them additionally susceptible to type II NK cells, rather than enhancing their killing by type I NK cells. This proposition would explain the lack of effect of acid treatment on the competing ability of tumour cells, when target cells are only lysed by type I NE cells.     

Modulation of natural cytotoxicity by alloantibodies: III. Augmentation of natural killer activity by alloantisera directed against K,D, and I regions of the murine H-2 complex

Natural killer activity of mouse spleen cells toward a human myeloid leukemia cell line, K562, can be enhanced by alloantisera directed against individual antigens in the H-2 region. By using a panel of 13 antisera (8 directed against antigens in the K and D regions and 5 directed against antigens in the I region) and four strains of mice (C57BL/6J, CBA, DBA/2, and A/J) it was found that certain antisera would stimulate target cell lysis by spleen cells only if the antisera had specificity for antigens which were a part of the haplotype represented on the spleen NK effector cells. Anti Ia antisera could stimulate the anti K562 NK activity of nude mouse spleen cells which lack mature T cells. Depletion of B cells and macrophages from nude spleen cells, by passing through a nylon-wool column also did not abolish the effect of anti-Ia antiserum. It appears likely therefore that the anti-Ia antibodies exert this effect directly on NK cells and that Ia antigens may be expressed on NK cells. Since the antisera directed against different antigens in H-2 complex irrespective of subregion specificity (K, D, or I) stimulated the NK activity of mouse spleen cells, the phenomenon offered an interesting method for testing the presence of a given alloantigen on mouse spleen cells. Log-dose response curves for the augmentation of lysis induced by appropriate alloantisera were linear over a dilution range of 1:320 to 1:5120. By using the dose-response curves, potency ratios of two preparations of antisera (directed against antigen 33 of the K region) could be successfully determined. Besides the K562 cell line, many human lymphoblastoid cell lines could also be used as target cells in this assay system.     

Phorbolester-treated human lymphocytes are susceptible to natural killer cell-mediated cytolysis

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was found in this study to render normal human lymphocytes susceptible to allogeneic and autologous natural killer (NK) cell lysis. Both T and non-T cells became susceptible after culture for 3 days in the presence of 1 ng/ml TPA. Effector cells were nonadherent mononuclear cells of low density, enriched for large granular lymphocytes and HNK-1+ cells. Activation of effector cells with interferon increased lysis of TPA-treated lymphocytes. The present system may provide a new model to study NK cell target structures.     

Interferon-gamma-treated K562 target cells distinguish functional NK cells from lymphokine-activated killer (LAK) cells

In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies.     

Products of the lipoxygenase pathway in human natural killer cell cytotoxicity

As earlier data suggested the importance of lipoxygenase activation for expression of human NK cell cytotoxicity, four different lipoxygenase inhibitors were tested for suppression of natural killer (NK) cell lysis. All inhibitors were found active at nontoxic concentrations with 50% inhibition at approximately 15 microM for nordihydroguaiaretic acid (NDGA). NK cell lysis could be reconstituted to NDGA-suppressed cells with leukotriene B4 (LTB4), the all-trans isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4, and 20-COOH-LTB4. LTB4 reconstitution was best in the concentration range 1-100 pM and near control levels at both higher and lower concentrations. Herpesvirus Ateles-transformed killer T cells could also be inhibited by NDGA. These data indicate that lipoxygenase activity is required for human NK cell lysis and that several different LTB4-related products can restore NK activity in inhibited cells; they also suggest that the lipoxygenase pathway is present in the killer cell population.     

Adriamycin induced resistance of sensitive K 562 cells to natural killer lymphocyte attack

Summary The effect of Adriamycin (ADM) on eryhtroleukaemia K 562 cell susceptibility to human natural killer (NK) cell activity has been studied. When cultivated for 3 days in the presence of 10 to 40 nM ADM, K 562 cells decreased their susceptibility to NK-mediated lysis in a dose-dependent fashion. At a concentration of 40 nM, previously found to induce optimal differentiation-associated properties in K 562 cells, the induced resistance to NK-mediated lysis increased progressively from day 1 to day 3 of culture. ADM treatment did not induce K 562 cells to release factors which interfered with NK activity since supernatants from ADM-treated K 562 cell cultures caused no significant modification in the NK lytic process. Binding to NK of ADM-treated K 562 cells was unaffected since treated and untreated cells had identical capacities in a conjugate-forming cell assay or adsorption of NK cells on target cell monolayers. In cold target competition assays ADM-treated K 562 cells acted as more effective competitors than untreated K 562 cells. These observations imply that the reduced killing of the ADM-treated K 562 cells was independent of target-NK cell recognition, and suggest that ADM treatment could allow malignant cells to escape NK surveillance.     

Human natural killer cells limit replication of herpes simplex virus type 1 in vitro

Studies were undertaken to determine whether natural killer (NK) cells could inhibit the replication of herpes simplex virus type 1 (HSV-1) in culture. In the absence of effector cells, HSV-1 was found to replicate in fibroblasts with up to a 100-fold increase in virus titer from 4 to 16 hr after incubation at 37 degrees C. Human peripheral blood mononuclear cells were found to limit virus replication in a dose-dependent manner, with the greatest inhibition being observed at the highest concentration evaluated: i.e., an effector:target ratio of 800:1. The antiviral effect was not observed when nonactivated or virus-activated mononuclear cells were added to the virus preparations at the end (instead of the beginning) of the assay period, indicating that the observed effect was not due to a nonspecific toxicity of soluble factors released from freeze-thawed effectors. Neither was inhibition of HSV-1 replication due to the generation of interferon (IFN) during the NK assay, because the addition of anti-IFN did not abrogate the antiviral effect. Thus, the inhibition of viral replication was most likely due to a cytotoxic effector rather than to release of soluble factors. The effector cells responsible for limiting HSV-1 replication were shown to be NK cells by a number of criteria. Mononuclear cells from both HSV-1 seropositive and seronegative donors limited virus replication; their activity could be boosted by pretreatment of effector cells with IFN; the effector cells which limited virus replication were found in Percoll gradient fractions enriched for large granular lymphocytes; and the effector cells shared the cell surface phenotype of NK cells--they were enriched in populations depleted of T cells by panning with Leu-4 and were depleted of activity by treatment with the anti-NK antibody Leu-11b plus complement. We conclude that human NK cells are capable of recognizing and lysing HSV-1-infected target cells before infectious virus progeny are generated. These results suggest that NK cells, acting early in the course of an infection, might serve to limit HSV-1 replication and therefore reduce the virus load in the host before the development of the adaptive immune response and clearance of the infection.     

Effects of adriamycin on the activity of mouse natural killer cells

Adriamycin, a widely employed anti-neoplastic agent, was found to have either inhibitory or stimulatory effects on NK activity, depending on the site examined. A single i.p. administration of ADM resulted in a rapid increase of cytolytic activity by PEC of various mouse strains. The effector cells appeared to be NK cells, being nonadherent and nonphagocytic; they expressed low amounts of Thy 1.2 antigen and had the same pattern of specificity as splenic NK cells. In contrast to the stimulatory effects of NK activity of PEC, ADM caused a transient dose-dependent depression of NK activity in the spleen, with a peak reduction at day 3 and recovery within a few days thereafter. The depressed NK activity could be reversed by removal of adherent cells by passage through a nylon column. Moreover, ADM induced cytostatic activity against tumor cells by macrophages, suggesting that activated macrophages may be responsible for suppression of splenic NK activity. The possible modulation of the levels of NK activity by ADM-induced macrophages was supported by mixture experiments, in which plastic adherent spleen cells from ADM-treated mice, but not from normal mice, inhibited the NK activity of normal spleen cells.