Antineoplastic effects of natural killer cells sorted with flow cytometry on brain tumors

For the purpose of FCM, we have developed a new method of sterile sorting of NK cells by negative selection using FITC-labelled monoclonal antibodies. In this method, over 95% lymphocytes with large granular lymphocytes (NK cells) were obtained by exclusion of Leu-1 and HLA-DR positive cells from human peripheral blood. From the results of cytotoxicity test of NK cells against 4 types of glioma cell lines and 6 kinds of cultured cells from surgical materials, the antineoplastic effect was recognized in 4 out of 6 surgical materials. Administration of NK cells through an Ommaya tube in 2 brain tumor patients caused no side effects. This technique is clinically applicable because 1) NK cells are easy to obtain, 2) NK cells show antineoplastic effect on brain tumors, and 3) the sensitivity can be evaluated in individual patients.     

Analysis of the murine lymphokine-activated killer (LAK) cell phenomenon: dissection of effectors and progenitors into NK- and T-like cells

Murine as well as human lymphokine-activated killer (LAK) cells have been reported to have several characteristics of T lymphocytes and to be clearly distinct from natural killer (NK) cells. The present study of murine LAK cells showed that cytotoxic cells generated in the presence of interleukin 2 IL 2 were heterogeneous with respect to cell surface markers of progenitor as well as effector cells. Negative selection of cells with antibodies and complement or positive selection by fluorescence-activated cell sorting unequivocally showed that LAK effector cells consisted of at least two clearly distinct populations, the relative contribution of which was dependent on donor organ and target cells studied. Approximately 40% of the cytotoxic activity of spleen-derived effector cells active against the NK-resistant targets EL-4 or MCA-5 was eliminated by treatment with antibodies to the NK-markers asialo-GM1 and NK 1 (NK-LAK). Approximately 60% of cytotoxic activity was associated with cells expressing the T cell marker Lyt-2, lacked NK 1, and was lacking or expressed only small amounts asialo-GM1 (T-LAK). The NK-LAK cells were of greater importance for the cytotoxic activity against the standard NK target YAC-1, although T-LAK cells also excerted significant cytotoxicity against this cell line. Limiting dilution analysis estimated that the minimal frequency of precursors developing into cells with cytotoxic activity against EL-4 was 1/6700 in spleen and 1/4200 in peripheral blood. The frequency of cells developing into cytotoxic effectors against YAC-1 cells was 1/3700 and 1/1450 in spleen and peripheral blood, respectively. Depletion of progenitor cells from spleen or peripheral blood expressing NK 1 or Lyt-2 by treating the cells with antibodies to these structures and complement indicated that NK-1-expressing cells were the dominating progenitor of the LAK cells irrespective of target cells used. Culture of murine lymphoid cells from spleen or peripheral blood with high concentrations of IL 2 results in the emergence of two different killer cell populations with phenotypic similarities to NK and T cells, respectively, both being able to kill targets resistant to resting NK cells. In contrast to numerous earlier reports, we concluded that LAK cells are heterogeneous with respect to surface markers, with a major population of LAK cells apparently representing IL 2-activated cells expressing cell surface markers associated with NK cells.     

Activation through CD3 molecule leads to clonal expansion of all human peripheral blood T lymphocytes: functional analysis of clonally expanded cells

Monoclonal antibodies directed against CD3, a T cell-specific surface molecule essentially required for activation of these cells, are highly mitogenic for resting human peripheral blood T lymphocytes. A predetermined optimal concentration of anti-CD3 monoclonal antibody WT32 was employed to activate T cells cultured in limiting-dilution microcultures containing irradiated feeder cells and exogeneous interleukin 2. Frequencies of cells triggered into clonal expansion by WT32 under these culture conditions were 0.57 to 0.72 and 0.90 to 1.10 in peripheral blood mononuclear cells and E rosette-positive cells, respectively. It appeared that WT32 could induce virtually every human peripheral blood T lymphocyte to expand into a clonal progeny of 5 to 40 X 10(4) cells in 14 to 18 days of culture. This progeny was tested for cytolytic effector function with 51Cr-labeled murine P815 targets in the presence of PHA to detect all cytotoxic T lymphocytes (CTL) regardless of specificity, and was also assayed for natural killer like activity against K562 target cells. Frequencies of cells in the human peripheral blood T cell compartment giving rise to a clonal progeny expressing CTL function was 1/3, whereas 1/6 to 1/5 expanded into effector cell populations possessing NK activity. Frequency analysis of CD4-positive and CD8-positive populations, activated by WT32 in limiting dilution microcultures, demonstrated that 1 to 6% of the CD4-positive and 100% of the CD8-positive peripheral blood T lymphocytes expanded into CTL.     

Phenotypic characterization of cynomolgus monkey natural killer cells

Natural killer (NK) activity of cynomolgus monkey peripheral blood lymphocytes (PBL) was determined using B95-8 cells as target cells. Examination for the reactivity of human NK-related monoclonal antibodies (mAbs), anti-Leu-7, anti-Leu-11b, anti-NKH1A, and NC-1, with cynomolgus PBL revealed that Leu-11b (CD16) was the only antigen expressed on cynomolgus PBL. The percentage of Leu-11b-positive (Leu-11b+) cells correlated well with the level of NK activity when PBL taken from 21 monkeys were tested. After depletion of Fc receptor-positive (FcR+) cells, NK activity was lost concomitantly with the disappearance of Leu-11b+ cells. These results show that cynomolgus NK cells are mainly FcR+ which can be detected by mAb directed to Leu-11b. Cynomolgus PBL were separated by Ficoll-Hypaque centrifugation after E rosette formation with 2-aminoethylisothiouronium bromide-treated sheep red blood cells, and NK activities of both E rosette-forming (E+) and nonforming (E-) fractions were determined. The high level of killing was observed in the E- fraction, suggesting that the majority of cynomolgus NK cells was contained in the E- fraction. The separation of PBL by Percoll discontinuous density gradient showed cynomolgus NK cells were enriched in the low density fractions.     

3-Carboxy-6-chloro-7-hydroxycoumarin: A highly fluorescent, water-soluble violet-excitable dye for cell analysis

In our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (∼0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays. Additional studies were performed, with BD Horizon V450-antibody conjugates being included in eight-color cocktails aimed at subsetting lymphocytes and myeloid cells. Such cocktails can frequently be unstable due to the tendency of one or more components to lose structural integrity, photobleach, or develop unwanted affinities for another component. However, the cocktails employed in this study enabled several different applications to be run and established that multicolor reagent mixtures containing V450-antibody conjugates are functional and stable.     

Natural killer cells in the blood and bone marrow of the rhesus monkey

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.     

Preferential elimination of NK and CTL functions by anti-D44 monoclonal antibody

Monoclonal antibodies reactive with T cells at various stages of maturation were used in negative selection experiments to study their effects on NK function in the presence of complement. Anti-D47 and anti-A50, respectively, directed against corticothymocytes and mature peripheral E+ cells were without effect. Anti-D66 reactive with an epitope of the T cell E receptor inhibited up to 60% of NK activity. Anti-D44, which primarily recognizes corticothymocytes and 60 to 80% of the E(+)-PBL was found to abrogate NK activity together with alloreactive CTL reactivity but to leave intact most of the MLR and PHA proliferative responses. Therefore D44 appears as a discrete antigen allowing preferential elimination of NK cells and CTL from PBL.     

A leukocyte subset bearing HLA-DR antigens is responsible for in vitro alpha interferon production in response to viruses

In this report, we characterize the major human peripheral blood nonadherent mononuclear cell subset that is responsible for the production in vitro of alpha-interferon (alpha IFN) in response to influenza, Sendai, and Newcastle disease viruses. Using a panel of anti-monocyte, anti-B, anti-T and anti-natural killer cell monoclonal antibodies to purify and recover both positive and negative cell populations, we show that the major alpha IFN-producing cells are HLA-DR(+) cells with no other surface markers characteristic of either lymphocytes or myelomonocytic cells. These cells copurify, on Percoll density gradients, with cells mediating NK activity, but the cell population responsible for alpha IFN production can be distinguished unambiguously from NK cells based on the former's reactivity with an anti-HLA-DR monoclonal antibody, the lack of reactivity with antibodies that detect the low-affinity receptor for IgG on human natural killer cells and granulocytes, and the inability to mediate spontaneous cytotoxicity. Double immunofluorescence assays indicate that cells of this subset, the lineage of which is as yet undetermined but which might be related to dendritic cells, constitute a minor proportion (approximately 1-1.5%) of the nonadherent mononuclear cells from healthy donors.     

Highly purified selective isolation of eosinophils from human peripheral blood by negative immunomagnetic selection

Eosinophils are a minority constituent in human peripheral blood. The study of eosinophils has been limited by difficulty in achieving sufficient cell number and purity. We describe a modified protocol for immunomagnetic cell separation for efficient isolation of human peripheral blood eosinophils. We employ a mixture of monoclonal antibodies (mAbs) directed against cell-surface antigens on human hematopoietic cells combined with secondary labeling with a colloidal suspension of magnetic dextran-iron particles for negative selection of eosinophils. Unwanted labeled cells are retained in the magnetized column, permitting high recovery (70%) and purity (>98%) of eosinophils while retaining cell viability. Eosinophils remain quiescent after isolation, and stimulation caused by cytokines upregulates (i) cell-cell or cell-extracellular matrix protein adhesion, (ii) secretion of bioactive mediators and (iii) cell-surface adhesion molecules. This method for purified isolation is accomplished in < or = 4 h and preserves eosinophils in a quiescent, viable state.     

Human neuroblastoma cell lines are susceptible to lysis by natural killer cells but not by cytotoxic T lymphocytes

The susceptibility of human neuroblastoma cells to direct cellular cytotoxicity has not been previously established. This is of particular interest because of their aggressive growth and low HLA expression. Neuroblastoma lines CHP 100 and CHP 126 were found to be excellent targets in 4-hr CML assays. Natural killer (NK) cells from fresh PBL and from an NK clone, 3.3, have high lytic activity against both cell lines. We also studied mixed lymphocyte culture-generated cytotoxic lines containing allo-specific cytotoxic T lymphocytes (CTL) directed against HLA antigens present on the neuroblastoma target cell lines. These lines did show excellent lytic activity, but cold target competition studies indicated that all of the lysis resulted from NK activity. This was verified by using inhibition studies with the use of monoclonal antibodies. OKT 3 and anti-HLA antibodies that block CTL function caused no reduction in kill. In contrast, anti-lymphocyte function antigen-1 (anti-LFA-1), which blocks both NK and CTL function, significantly inhibited lysis. These results serve as a functional confirmation of earlier findings of a very weak expression of HLA-A,B,C and beta 2-microglobulin on neuroblastoma cells.     

Role of IgE receptors in effector function of human eosinophils

After analysis of the technical parameters of the rosette assay with human IgE-coated erythrocytes, Fc epsilon receptors for IgE (Fc epsilon R) on human peripheral blood eosinophils were compared to Fc epsilon R on lymphocytes and monocytes. Antibodies directed against Fc epsilon R on lymphocytes and monocytes inhibited the IgE rosettes formed by eosinophils from hypereosinophilic patients, which suggests that Fc epsilon R on eosinophils were antigenically related to Fc epsilon R on lymphocytes and monocytes. Fc epsilon R on human eosinophils were shown to participate in the killing effect of Schistosoma mansoni schistosomula in vitro in the presence of purified eosinophils from highly hypereosinophilic patients (blood counts greater than 3000/mm3) and anti-schistosomula IgE antibodies present in S. mansoni-infected patient sera. Similar levels of inhibition of cytotoxicity were obtained after preincubation of eosinophils with aggregated human IgE or with anti-Fc epsilon R antibodies, whereas preincubation with aggregated IgG or with anti-C3b receptor antibodies did not decrease the killing effect for schistosomula targets. This IgE-dependent cytotoxic capacity seemed restricted to eosinophils with an abnormally low density ("hypodense" cells) present only in highly hypereosinophilic patients. These observations might be related to nonparasitic situations in which increased levels of IgE and tissue or blood eosinophils are observed.     

Functional characterization of LFA-1 antigens in the interaction of human NK clones and target cells

In the present studies we analyzed the role of LFA-1 antigens in the interaction between NK clones and target cells. The use of various cloned NK cell lines allowed us to analyze homogeneous populations of NK cells which ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotype and specificity. Indirect immunofluorescence with monoclonal antibodies against the alpha (MHM24) and beta (MHM23) chains of the LFA-1 antigen revealed similar patterns of positive reactivity with all NK clones. Both monoclonal antibodies exerted a significant blocking effect on NK cytotoxicity against target cells such as Molt-4 and CEM, whereas the inhibition was very weak against other targets such as K562 and HSB cells. Additive blocking effects were seen when both monoclonal antibodies MHM23 and MHM24 were added to the cytotoxicity assays. When we compared the inhibitory effect of MHM23 and MHM24 on uncultured peripheral blood NK cells and IL 2-activated NK cells, inhibition of cytotoxicity also was found to be primarily dependent on the individual target cells. Thus, the inhibitory activity of anti-LFA-1 antibody was shown to be independent of the phenotypic and functional heterogeneity of the NK clones, activated NK cells, and unstimulated NK cells utilized in these studies. These blocking effects were found to be independent of the LFA-1 antigen expression on the target cell membrane and inhibition occurred only when antibody was bound to the effector cells. Comparison of the effects of anti-LFA-1, anti-T3, and anti-clonotypic antibodies against a Ti-like structure of different NK clones with a mature T cell phenotype demonstrated that each of these antibodies acts on the effector cells in an independent and additive fashion. However, unlike T3 and NKTa antigen, LFA-1 antigen expression is not modulated by cell surface interaction with antibodies specific for this molecule.     

T lymphocyte-mediated cytotoxicity against autologous EBV-genome-bearing B cells

We have stimulated human peripheral blood lymphocytes in vitro with autologous EBV-infected or noninfected B cells. A cytotoxic response was obtained only when virally infected cells were used. The activity of the effector cells was restricted by the major histocompatibility complex and was directed against EBV-genome-bearing targets. The highest cytolytic response was obtained when lymphocytes of individuals previously exposed to the virus (EBV-VCA positive) were used. Lymphocytes of noninfected donors (EBV-VCA negative) gave a low response; the relative frequency of their effector cells was at least 4-fold lower. Lymphocytes of newborns did not respond. The cytotoxic activity was mediated by T lymphocytes of the cytotoxic/suppressor subset, as determined by cytofluorographic analysis and antibody plus complement-mediated lysis, using monoclonal antibodies to human lymphocyte surface antigen.     

Constitutive release of IL6 by human papillomavirus type 16 (HPV16)-harboring keratinocytes: a mechanism augmenting the NK-cell-mediated lysis of HPV-bearing neoplastic cells.

In the present study we demonstrate that the cultured human keratinocyte cell line (SK-v cells) harboring and expressing integrated human papillomavirus type 16 (HPV16) DNA sequences constitutively releases IL6, which is known as a pleiotropic immunoregulatory cytokine of potential antitumor properties. The presence of IL6 activity in SK-v cell-conditioned media (SK-v CM) was demonstrated by tritiated thymidine incorporation into IL6-dependent B9 murine plasmacytoma cells. The effect on B9 cells was specific since it could be inhibited by anti-IL6 neutralizing antibodies but not by a normal control serum. IL6 did not affect SK-v cell growth; however, it significantly augmented NK cell activity of human peripheral blood lymphocytes against both K562 erytholeukemic and SK-v cells as assessed by 51Cr release assay. SK-v CM displayed NK cell-augmenting activity that copurified with IL6 activity in both size exclusion and anion-exchange HPLC. Furthermore, SK-v cell-derived NK cell stimulatory activity could be neutralized with anti-IL6 antibodies. These results suggest that HPV-harboring neoplastic cells can release IL6 which may indirectly mediate tumor death by augmentation of NK cell activity.     

Human trophoblast cell resistance to decidual NK lysis is due to lack of NK target structure.

We have previously shown that human cultured trophoblast cells are resistant to lysis by natural killer (NK) cells from both peripheral blood and decidua although cells are present in decidua which do exhibit NK activity against K562(1). Using a cold-target inhibition assay and a single-cell conjugate assay we have now examined whether these trophoblast cells have NK target structures on their surfaces. Our findings indicate that first-trimester human trophoblast cells do not express surface structures recognized by decidual Leu19+ (CD56+) large granular lymphocytes (LGLs) isolated from human decidua. Immunostaining of the conjugates formed between decidual NK effectors and K562 cells confirmed that these effector cells are CD56+ LGLs.     

Phorbol ester-induced lymphocyte adherence: selective action on NK cells

Treatment of human peripheral blood lymphocytes (PBL) with phorbol dibutyrate (PDBU) for 20 to 45 min at 37 degrees C induces adherence of 5 to 30% of the cells to plastic. The adherent cells (pAd) were highly enriched in NK cells on the basis of the following findings: 1) they exhibit high NK and ADCC activity but do not lyse the NK-resistant cell line, Daudi; 2) cytotoxic activity is enhanced by pretreatment with interferon-alpha (IFN-alpha); 3) the surface markers of these cells, as determined with monoclonal antibodies, are consistent with NK cells; and 4) they are enriched with cells morphologically similar to large granular lymphocytes. Conversely, the PDBU-nonadherent cells were substantially depleted of NK cells. The fact that the pAd cells do not lyse the Daudi line and that their NK activity can be further augmented by IFN-alpha would suggest that the pAd are enriched for NK cells rather than changed in their characteristics as a result of the separation procedure. Moreover, no consistent and appreciable modulation of NK activity induced by PDBU was observed. This report therefore demonstrates that PDBU selectively induces NK adherence of NK cells, which may have practical as well as biological implications.     

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.     

Antibody 3G8, specific for the human neutrophil Fc receptor, reacts with natural killer cells

Antibody 3G8 reacts with the receptor for the Fc fragment of aggregated IgG present on the majority of neutrophilic granulocytes and on a small proportion of lymphocytes. In this report, we compare the pattern of reactivity of antibody 3G8 on peripheral blood lymphocytes (PBL) and on polymorphonuclear leukocytes (PMN) with that of antibody B73.1, which reacts with the Fc receptor of natural killer (NK) cells or with a molecule functionally associated with it. We show that 3G8 reacts with the same PBL subset detected by antibody B73.1 and is responsible for virtually all NK cytotoxic activity. The lymphocyte subset recognized by the two antibodies has the morphology of large granular lymphocytes and includes neither B nor T cells. Our results indicate that NK cells and PMN express the same Fc receptor for immune complexes, and that B73.1 and 3G8 recognize on the same receptor two distinct epitopes that are preferentially expressed on NK cells and on PMN, respectively.