Analysis of human V alpha 24+ CD4+ NKT cells activated by alpha-glycosylceramide-pulsed monocyte-derived dendritic cells

Human V alpha 24+ NKT cells with an invariant TCR (V alpha 24-J alpha Q) have been shown to be specifically activated by synthetic glycolipids such as alpha-galactosylceramide and alpha-glucosylceramide in a CD1d-restricted and V alpha 24 TCR-mediated manner. We recently characterized V alpha 24+ CD4- CD8- double negative (DN) NKT cells using alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. Here, we compare V alpha 24+ CD4+ NKT cells with human V alpha 24+ DN NKT cells from the same donor using alpha-galactosylceramide-pulsed monocyte-derived dendritic cells. Human V alpha 24+ CD4+ NKT cells were phenotypically and functionally similar to the human V alpha 24+ DN NKT cells characterized previously. Both of them use V alpha 24-J alpha Q-V beta 11 TCR and express CD161 (NKR-P1A), but not the other NK receptors tested so far. They also produce cytokines such as IL-4 and IFN-gamma, and, in regard to IL-4 production, V alpha 24+ CD4+ NKT cells produce more IL-4 than V alpha 24+ DN NKT cells. The cells exhibit marked cytotoxic activity against the U937 tumor cell line, but not against the NK target cell line, K562. Although at least some of the factors responsible for the stimulation of V alpha 24+ NKT cells have been clarified, little is known regarding the killing phase of these cells. Here we show that the cytotoxic activity of V alpha 24+ NKT cells against U937 cells is mediated mainly through the perforin pathway and that ICAM-1/LFA-1 as well as CD44/hyaluronic acid interactions are important for the effector phase of V alpha 24+ NKT cell-mediated cytotoxicity against U937 cells.     

Human monocyte-mediated tumor cytotoxicity. I. Demonstration of an oxygen-dependent myeloperoxidase-independent mechanism

The cytolytic capacity of monocytes per se and stimulated monocytes has been documented to only a limited extent, and when observed has been ascribed to the generation of a variety of cytolytic molecular entities. In the present study we have examined de novo human monocyte-mediated tumor cytotoxicity and that induced by the agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Cytolytic function was analyzed by reference to the release of [111In] oxine from two prelabeled tumor cell lines, K562 and U937, in a 16-hr assay in the presence of serum to more closely mimic in vivo circumstances. Observed cytolysis was clearly related to TPA concentration and effector cell number. Maximal cytolysis was obtained with TPA at 5 ng/ml, at which specific releases were 43% +/- 6 and 18% +/- 5 (mean +/- 1 SEM) at an effector cell to target cell (E:T) ratio of 2.5:1 and 65% +/- 6, and 41% +/- 12 at an E:T ratio of 20:1, for K562 and U937, respectively. In contrast, unstimulated monocytes expressed minimal cytolytic activity, or at best a low cytotoxic effect at high cellular ratios. When TPA-stimulated monocyte-mediated cytolysis was examined, catalase (2750 U/ml) inhibited K562 and U937 cytolysis by 92% and 84%, respectively; superoxide dismutase (300 U/ml) only inhibited cytotoxicity by 17% and 24%, respectively, implicating a central role of H2O2 rather than superoxide ions. Sodium azide (1 mM), an inhibitor of myeloperoxidase, did not diminish cytolysis; in contrast, it increased K562 and U937 cytolysis by 34% and 57%. This increased cytotoxicity was observed for K562 at low levels of cytotoxicity. These data tend to dismiss an essential role of the H2O2-halide-myeloperoxidase pathway of cytolysis. The OH scavengers, histidine (20 mM) and ethanol (40 mM), did not affect K562 killing; mannitol (50 mM), another OH scavenger, had only a slight inhibitory effect (23%). Finally, H2O2 generated by a glucose-glucose oxidase system directly mediated K562 killing and, to a lesser extent, U937 lysis. These results point strongly towards the role of: 1) a myeloperoxidase-independent mechanism of cytotoxicity, with 2) H2O2 as a key mediator of the cytolytic mechanism, and 3) a limited role of O2.- in synergy with H2O2 in the cytolytic activity of monocytes, and suggest that significant cytolytic function requires an inductive event.     

Tumor necrosis factor-alpha dependent cytotoxicity of human skin mast cells is enhanced by anti-IgE antibodies

Mast cells dispersed from human skin and purified by density-gradient centrifugation were cytotoxic toward the mouse fibrosarcoma cell line WEHI-164. Skin mast cells were not cytotoxic toward the NK cell-sensitive cell line K562. Killing of WEHI-164 occurred over a prolonged (greater than 18 h) period of incubation with mast cells and was effectively inhibited by polyclonal antibodies and mAb against TNF-alpha suggesting that this cytokine plays an important role in mast cell-mediated cytotoxicity. Whereas lysates of rat peritoneal mast cells exhibited cytotoxicity toward WEHI-164, this was not found with lysates of unstimulated skin mast cells suggesting that TNF-alpha is not stored preformed in the latter. Killing of WEHI-164 cells by skin mast cells was enhanced by anti-IgE and there was a significant correlation between histamine release and cytotoxicity after activation with this stimulus. We conclude that human skin mast cells are a potential source of TNF-alpha and suggest that these cells, particularly after activation, might contribute to the synthesis of this multifunctional cytokine in inflammatory sites.     

Bcr-abl translocation can occur during the induction of multidrug resistance and confers apoptosis resistance on myeloid leukemic cell lines

Apoptosis was studied in parental and mdr-1 expressing U937, HL60 and K562 myeloid leukemic cell lines using mdr unrelated inducers of apoptosis such as Ara-C, cycloheximide, serum deprivation, ceramide, monensin and UV irradiation. Apoptosis was efficiently induced by all these treatments in U937 and HL60 cells while K562 cells exhibited an apoptosis-resistant phenotype except with UV and monensin. The pattern of apoptosis resistance in mdr-1 expressing U937 (U937-DR) and HL60 (HL60-DR100) was similar to that presented by K562. This apoptosis-resistant phenotype of mdr cells was not overcome by concentrations of verapamil inhibiting the P-gp 170 pump. The acquisition of this phenotype was posterior to the mdr-1 expressing phenotype since a HL60-DR5 variant, selected at the beginning of the induction of resistance, presented a low level of mdr-1 expression without resistance to apoptosis. The variations observed in the Fas (CD95) expression between sensitive and resistant cells were not sufficient to account for apoptosis resistance. However, a high expression in Abl antigen was found in all the apoptosis-resistant cells. RT-PCR and Western blot analysis showed that this increase in Abl antigen content was accompanied by the expression in U937-DR and HL60-DR100 cells of a hybrid bcr/abl mRNA and a 210 kD Bcr/Abl protein which was constitutive in K562. This expression was due to the translocation of abl and the amplification of the bcr-abl translocated gene. These results are in agreement with the role of Bcr/Abl tyrosine protein kinase as an inhibitor of apoptosis independently of the mdr-1 expression. They also suggest that translocation of the abl gene in the bcr region is a highly probable rearrangement in the mdr-1 expressing myeloid cells and that Bcr/Abl tyrosine kinase effect on apoptosis needs the regulation of intracellular pH and is inactive against UV-induced apoptosis.     

The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing

The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.     

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.     

Modulation of natural killer-mediated lysis by red blood cells

Natural killer (NK)-mediated cytotoxicity is significantly enhanced in the presence of red blood cells (RBC). The enhancement is dose dependent on the number of RBC present and can be induced by autochthonous, allogeneic, or xenogeneic RBC. The enhancement was demonstrated in cytotoxicity against two different NK-sensitive tumor target cell lines, K562 and U937, and by three different assay systems, chromium release, lactate dehydrogenase release, and inhibition of thymidine incorporation. RBC directly enhance the cytotoxic activity of NKH-1/Leu19+ large granular lymphocyte NK cells. Intact RBC have to be present during the cytotoxicity assay to induce the enhancement, which probably occurs at a postbinding, preprogramming phase. The anti-CD2 antibody Leu5 cannot block the enhancement at a concentration inhibitory to lymphocyte rosetting with sheep RBC, suggesting that the enhancement is not induced by interaction through the CD2 antigen. These results indicate that RBC are a potent modulator of NK cytotoxicity and suggest that in vitro NK studies using purified lymphocytes may not always truly reflect NK activity in the blood stream.     

Binding of sialyl Lewis X antigen to lectin-like receptors on NK cells induces cytotoxicity and tyrosine phosphorylation of a 17-kDa protein

BACKGROUND: Natural killer (NK) cells mediate cytotoxicity through cell-surface receptors including lectin-like receptors. We have investigated whether sialyl Lewis X (sLe(X)) antigen, Neu5Acalpha2,3Galbeta1,4(Fucalpha1,3) GlcNAc-R, can bind to the lectin-like receptors on human NK-derived KHYG cells, using transferrin secreted by human hepatoma-derived HepG2 cells (Hep-TF), whose N-glycans are rich in alpha1,3-fucosylated bi-, tri-, and tetra-antennary type complexes, and commercially available human transferrin (Nor-TF), which is comprised of bi-antennary N-glycans without alpha1,3-fucosylation. RESULTS: High sLeX-expressing erythroleukemia-derived K562 cells isolated from fucosyltransferase-3-transfected cells were 2.5-fold more susceptible than wild-type K562 cells to KHYG cells. Fluorescein isothiocyanate (FITC)-labeled Hep-TF bound 1.8-fold more strongly to KHYG cells than did FITC-labeled Nor-TF; the binding was suppressed by treatment with anti-NKG2D, anti-NKG2C, anti-CD94 and anti-CD161 antibodies. FITC-labeled Hep-TF bound more strongly to human monocyte-derived U937 cells transfected with NKG2D and CD94 than to wild-type U937 cells. Moreover, tyrosine phosphorylation of a 17-kDa protein in the KHYG cells was enhanced by incubation on a Hep-TF coated plate and treatment with an anti-NKG2D antibody, but not by a Nor-TF coated plate and an anti-CD94 antibody. CONCLUSION: The interaction of sLe(X) antigen with lectin-like receptors on NK cells induces cytotoxicity that is mediated through a tyrosine-phosphorylated 17-kDa protein.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

Expression of glycosylphosphatidylinositol-anchored CD59 on target cells enhances human NK cell-mediated cytotoxicity

NK cell-mediated cytotoxicity of target cells is the result of a balance between the activating and inhibitory signals provided by their respective ligand-receptor interactions. In our current study, we have investigated the significance of CD59 on human target cells in modulating this process. A range of CD59 site-specific Abs were used in NK cytotoxicity blocking studies against the CD59-expressing K562 target cell line. Significantly reduced cytotoxicity was observed in the presence of Abs previously shown to lack blocking capacity for C-mediated lysis. We investigated the consequences for alternative membrane attachment modalities, namely bis-myristoylated-peptidyl (BiMP) and GPI anchoring, on CD59-negative U937 cells. Expression of GPI-anchored CD59 either via transfection or incorporation rendered U937 targets more susceptible to NK cytotoxicity, whereas incorporation of CD59 via a BiMP anchor to similar levels did not alter susceptibility to NK cytotoxicity. Localization of both BiMP- and GPI-anchored CD59 proteins was shown to be within the lipid raft microdomain. A role for the GPI anchor and independence from glycosylation status was confirmed by expression of transmembrane-anchored CD59 or unglycosylated CD59 and by testing in NK cytotoxicity assays. To investigate mechanisms, we compared the signaling capacity of the various forms of expressed and incorporated CD59 following Ab cross-linking in calcium flux assays. GPI-anchored CD59, with or without glycosylation, mediated activation events, whereas CD59 forms lacking the GPI anchor did not. The data show that the increased susceptibility of target cells expressing CD59 to NK cytotoxicity requires GPI anchor-mediating signaling events, likely mediated by interactions between GPI-anchored CD59 on targets and NK receptors.     

Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.     

Protective activity of adult T cell leukemia-derived factor (ADF) against tumor necrosis factor-dependent cytotoxicity on U937 cells.

Adult T cell leukemia-derived factor (ADF) is a human homologue of thioredoxin with many biologic functions including IL-2R induction, growth promotion, thiol-dependent reducing activity, and radical scavenging activity. The regulatory effect of ADF on the cytotoxic activity of TNF was examined by using a human histiocytic lymphoma cell line, U937. When U937 cells were preincubated with recombinant ADF (rADF) (0.1-100 micrograms/ml) at 37 degrees C for 30 min, TNF-dependent cytotoxicity on U937 cells was markedly inhibited. This inhibitory effect was as high as 95% in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (rADF 100 micrograms/ml) and 85% in the 51Cr-releasing assay (rADF 10 micrograms/ml). After pretreatment of U937 cells with IFN-gamma to augment the sensitivity to TNF, an inhibitory effect of rADF was also found. When U937 cells were washed after preincubation with rADF, resistance to TNF-dependent cytotoxicity was still observed, indicating that rADF inhibited the sensitivity of U937 to TNF-dependent cytotoxicity rather than modifying TNF molecules. Scatchard analysis of TNF receptors on U937 cells using 125I-TNF showed that rADF modulated neither the density nor the affinity of the cell membrane significantly. rADF also reduced the cytotoxicity induced by anti-Fas IgM mAb which shows cytotoxicity quite similar to TNF. rADF (10 micrograms/ml) reduced 90% of the cytotoxicity by anti-Fas IgM mAb, without a detectable change either in Fas Ag expression (MFI 58.1 vs 53.3) or in the degradation of anti-Fas IgM mAb as determined by flow cytometric analysis. These findings indicated that the rADF-induced resistance to the cytotoxic effect of TNF and anti-Fas mAb was not related to the modulation of the TNF receptor or Fas Ag.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells

Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C(6)-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.     

Hybrid molecules based on fullerene C60 and 5Z,9Z-dienoic acids: Synthesis and cytotoxic activity

The present research project details synthesis of new hybrid methanofullerenes based on acetylene and triazole esters of malonic acid containing 5Z,9Z-dienoic acids and fullerene C₆₀ under Bingel-Hirsch conditions, including study of the cytotoxic activity with respect to Jurkat, K562, U937 and HL60 tumor cell lines. Hybrid methanofullerenes containing acetylenic fragments, unlike triazole substituents, were found to exhibit higher cytotoxicity, but are characterized by lower selectivity of action in relation to healthy cells.     

β1 Integrin triggering affects leukemic cell line sensitivity to natural killer cells

The role of beta1 (CD29) integrins in natural killer (NK) cell-target cell conjugation and cytotoxicity has not been clearly established. Ligation of beta1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the beta1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of beta1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-beta1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207-218 of the beta1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from beta1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-beta1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-beta1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-beta1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through beta1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity.     

Functional heterogeneity among herpes simplex virus-specific human CD4+ T cells.

One hundred thirteen HSV-specific CD4+ T cell clones were established from the PBL of a healthy person and their functional heterogeneity was investigated. All clones proliferated in response to stimulation with HSV in the presence of autologous APC. Among those, 48 clones showed cytotoxic activity to HSV-infected autologous EBV-transformed lymphoblastoid cell line, but not to HSV-infected autologous fibroblasts, HSV-infected allogeneic cells, or K562 cells (group 1). Five clones showed cytotoxicity against HSV-infected autologous cells as well as HSV-infected allogeneic cells and K562 cells (group 2). The cytotoxicity of these clones was found to be mediated by the direct killing but not by the "innocent bystander" killing of target cells. Sixty clones showed no cytotoxic activity, however, among these, 23 revealed HLA-unrestricted and nonspecific cytotoxicity in the presence of PHA in culture (group 3), and the remaining 37 did not show any cytotoxic activity even in the presence of PHA (group 4). The cytotoxic patterns of these clones did not change in activated and resting phases, suggesting that the difference in cytotoxic ability does not depend on cell cycles. The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR or anti-CD3 mAb to the culture, whereas these mAb had no effect on the cytotoxicity of group 2. All four groups of clones had helper activity for anti-HSV antibody production by autologous B cells. Moreover it was found that all groups of clones simultaneously produced IL-2, IL-4, and IFN-gamma after culture with APC followed by HSV Ag stimulation. The surface phenotype of all clones was uniformly CD2+, CD3+, CD4+, CD8-, CD29+, CD45RA-, but expression of Leu 8 was varied. These data therefore indicate that HSV-specific human CD4+ T cells are classified into at least four groups according to the presence and specificity of cytotoxicity, i.e., Th cells with HSV-specific and HLA-class II-restricted cytotoxicity, Th cells with HLA-unrestricted and nonspecific cytotoxicity, Th cells with lectin-dependent cytotoxicity, and Th cells without cytotoxic activity. The present finding of functional heterogeneity among virus-specific human CD4+ T cells might shed light on the pathogenesis of CD4+ T cell immunodeficiency, such as human retrovirus infections.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

Lymphokine release, suppressor cell generation, cell surface markers, and cytotoxic activity in cancer patients receiving natural interleukin-2

We monitored patients treated for 5 days with continuous infusion of increasing doses (3 to 6 x 10(6) U/d) of natural interleukin-2 (IL-2). CD16+, CD25+, and CD56+ cells increased after treatment. Plasma tumor necrosis factor-alpha (TNF-alpha) levels, but not interferon-gamma (IFN-gamma) levels, increased during IL-2 treatment, but spontaneous and IL-2-stimulated TNF-alpha secretion in vitro remained abnormally low. However, mitogen-stimulated TNF-alpha release was normal. Mitogen-stimulated, but not IL-2-stimulated, IFN-gamma release was strongly depressed. Low spontaneous and IL-2-stimulated cytotoxicity on K562 or Daudi increased after treatment. Low suppressor cell generation also normalized after treatment. This appears to be the first reported study of immunologic monitoring of cancer patients treated with natural rather than recombinant IL-2.