Cytotoxicity to tumor cells of monocytes from normal individuals and cancer patients

This study was aimed at characterizing the parameters which regulate human monocyte-mediated cytotoxicity to tumor cells as well as characterizing the target cell specificity, kinetics, etc., of the cytotoxic mechanism. Normal human peripheral blood monocytes were cytotoxic to tumor cells in an in vitro assay, measuring release of [³H]thymidine from human target cells. Monocyte cytolysis was observed with several adherent tumor lines including T24, a bladder cancer line; LR, a melanoma line; and an SV40-transformed W138 fibroblast line, with maximal cytolysis observed at 72 h. Lymphokines were not required to induce and only infrequently enhanced monocyte cytotoxicity. Prolonged exposure of monocytes to lymphokines or in vitro culturing of monocytes prior to lymphokine exposure did not alter the monocytes' response to lymphokine signals with respect to cytotoxicity. Lymphokines induced monocytes to exhibit enhanced spreading, suggesting that monocytes were susceptible to lymphokine signals but that the development of cytolytic function was independent of lymphokines. In contrast to the cytolysis of adherent tumor cells, monocytes were less effective in killing the non-adherent lymphoid target cells K562, Raji, and CEM. Monocytes were selectively cytotoxic to tumor cells and generally did not kill normal human fibroblast cell lines or PHA-stimulated lymphocytes. Monocytes from cancer patients exhibited normal cytotoxicity to several human tumor lines. Plasmas from some cancer patients were inhibitory to cytotoxicity mediated by both autologous monocytes and normal monocytes.     

Variables and specificity of in vitro lymphocyte-mediated cytotoxicity in human melanoma.

In vitro cell-mediated cytotoxicity (CMC) assays have been carried out in human melanoma system with blood effector lymphocytes on [3H]proline-labeled target cells in a 48-hr microcytotoxicity technique. Three lymphocyte purification procedures (Ficoll:Hypaque gradient, plasma gel sedimentation followed by nylon column incubation, and plasma gel sedimentation followed by separation with nylon powder and glass beads) are compared in parallel experiments for characteristic effector cell composition and cytotoxic potential against target cells of dissimilar histology. The cytotoxicity is defined by the loss of target cell 3H cpm as measured by residual target cell 3H cpm in individual microwell following incubation with lymphocytes. Target cell 3H cpm loss by test lymphocytes is compared with target cell 3H cpm loss by several age and sex matched control lymphocytes (from normal donors and unrelated cancer patients); further comparison between the various control lymphocytes is also made in each assay. As control for target cells, autologous fibroblasts and homologous tumor cells of dissimilar histology are always included in each assay. Specific cytotoxicity is defined as statistically significant and selective destruction of only melanoma cells by the test lymphocytes as compared to the control lymphocytes. Significant but nonselective destruction of 2 or more target cells of unrelated histology is regarded as nonspecific cytotoxicity, while no destruction of any target cells signifies no cytotoxicity. The Ficoll:Hypaque preparations consistently exhibit the highest nonlymphocytic cell contamination (8 to 16%); the nonlymphocytic cells are, almost exclusively, monocytes. They also produce relatively high percentage of thymus independent (B) cells (8 to 15%). The ultimate cell composition of the 2 plasma gel-nylon preparations is essentially identical. In either plasma gel-nylon preparations, the nonlymphocytic contamination is minimal (0 to 4%) and thymus-dependent (T) cell percentage is considerably higher (92 to 99%) with none or few B cells.     

Tumour-associated lymphocyte cytotoxicity superimposed on “Spontaneous” cytotoxicity in melanoma patients

Lymphocytes from 16 stage 1, 6 stage II and 31 stage III melanoma patients (MP) and 51 healthy donors (HD) were tested as far as possible in parallel on a melanoma cell line (NK1-4), a bladder carcinoma cell line (T 24) and 18 different short-term melanoma cultures. Lymphocytes from MP and HD showed cytotoxic effects towards all three types of target cells. Lymphocytes from HD showed the strongest “spontaneous” cytotoxic effects on NKI-4 cells whereas, in general, weak cytotoxic effects were seen on short-term cultured melanoma cells. Within the different lymphocyte donor groups an enormous variation in cytotoxic effects was observed. However, the overall cytotoxic effects of stage I and II MP were significantly higher than those of the HD-group. Stage I MP showed significantly stronger cytotoxic effects on NKI-4 cells than on T 24 cells, indicating that tumour-associated lymphocyte cytotoxicity was superimposed on spontaneous cytotoxicity.     

Comparison of recombinant tumor necrosis factor and the monocyte-derived cytotoxic factor involved in monocyte-mediated cytotoxicity

A direct comparison of recombinant tumor necrosis factor (rTNF) and the monocyte-derived cytotoxic factor (CF) which is involved in monocyte-mediated cytotoxicity revealed immunological, physiochemical, and biological similarities, indicating that TNF is an effector molecule in monocyte-mediated cytotoxicity. Neutralizing antiserum raised against rTNF completely inhibited the ability of CF-containing monocyte supernatants to induce cytolysis and cell death of sensitive target cells and, conversely, antiserum raised against purified CF completely inhibited the cytotoxic activity of rTNF. Both CF and rTNF have an apparent isoelectric point of 5.8-5.9 as determined by chromatofocusing, and a molecular weight of about 40,000 as determined by gel filtration. Moreover, when present in monocyte supernatants with a total protein concentration of about 1 mg/ml and 0.1% sodium dodecyl sulfate, both CF and rTNF migrated with a molecular weight of about 35,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pure rTNF, however, migrated with a molecular weight of 17,000, suggesting that the relative amount of sodium dodecyl sulfate to protein is critical for dissociating the apparent dimeric structure of TNF. CF and rTNF were also similar with respect to their ability to kill various types of target cells the sensitivity of which to TNF differ, and the dose-response curves of cytotoxicity obtained with CF-containing monocyte supernatants and rTNF were similar. As is the case with anti-CF serum, anti-rTNF serum inhibited drug-dependent cellular cytotoxicity and cytolysis mediated by both freshly isolated monocytes and in vitro cultured unactivated and lymphokine-lipopolysaccharide activated monocytes, indicating that TNF is an effector molecule in both drug-dependent cellular cytotoxicity and "classical" monocyte-mediated cytotoxicity.     

Induction of cytotoxic activity in human lymphocytes against autologous and allogeneic melanoma cells in vitro by culture with interleukin 2

The influence of interleukin 2 (IL2) on the cytotoxic activity of lymphocytes from patients with melanoma against autologous and a variety of allogeneic melanoma cells was studied. IL2 was produced from blood lymphocytes cultured for 24 h with phytohaemagglutinin (PHA) and purified by membrane chromatography to exclude PHA. Lymphocytes from 13 patients with melanoma at various clinical stages were cultured for 6 days with IL2 (2 U/ml) and then tested for cytotoxic activity against autologous melanoma cells, three allogeneic melanoma and three non-melanoma cells. Autologous cytotoxicity was generated by culture with IL2 alone and was not increased by culture with both IL2 and autologous tumour cells. Marked increases in cytotoxic activity were also generated against the allogeneic target cells and were maximal against the NK-insensitive Chang target cells. Similar degrees of cytotoxicity were induced by IL2 stimulation of lymphocytes from melanoma patients, patients with non-melanoma carcinoma and normal subjects against the allogeneic target cells. Cold target inhibition studies were carried out against IL2 induced autologous cytotoxicity in five patients. In four of five studies the autologous target cells inhibited more than the allogeneic target cells. There was no significant difference between the inhibition produced by allogeneic melanoma cells and that produced by non-melanoma cells. Similarly, in studies against allogeneic target cells, there was no significant difference in the inhibition produced by allogeneic melanoma compared to non-melanoma target cells. This applied irrespective of whether effector cells were from melanoma or non-melanoma subjects. These results suggest that lymphocytes from patients with melanoma are primed against autologous antigens in vivo and that provision of a second signal, IL2, in vitro can induce cytotoxicity against the autologous tumour. The cytotoxicity generated against the allogeneic target cells did not appear to have specificity to melanoma. Several results, such as the pattern of cytotoxicity against the target cells and changes in cell surface markers on the lymphocytes during culture, suggested that cytotoxicity was mediated by activated T cells rather than by natural killer cells. These findings appear to have important implications both in the understanding of tumour host relationships and for the use of IL2 in therapy.     

Natural cytotoxicity of human blood monocytes and natural killer cells and their cytotoxic factors: discriminating effects of actinomycin D

The effect of actinomycin D on target susceptibility to human blood natural killer (NK) cells and monocytes was analysed in direct cell-mediated and their cytotoxic factor-mediated cytotoxicity assays. Treatment of K562 cells with actinomycin D reduced their susceptibility to lysis by non-adherent lymphocytes and Percoll-purified large granular lymphocytes (LGL) in a 4-hr 51Cr-release assay, without affecting their sensitivity to monocytes purified by adherence to autologous serum-coated plastic surfaces. The drug treatment caused no shift in the kinetics of cytotoxicity. In the target binding assay LGL formed fewer conjugates with actinomycin-D-treated K562 cells than with untreated ones, while the binding of monocytes to targets was not reduced by the drug treatment of K562 cells. The cold target competition assay revealed that actinomycin-D-treated cold K562 cells showed less successful inhibition than untreated cold K562 cells. Lymphocytes and monocytes could be induced to release soluble cytotoxic factors, termed natural killer cytotoxic factors (NKCF) and monocyte cytotoxic factors (MCF), respectively, when co-cultured with K562 cells. Both cytotoxic factors lysed NK-sensitive target cells in a 48-hr assay. Actinomycin-D-treated K562 cells reduced or abolished the ability to stimulate the release of NKCF from lymphocytes, whereas they induced MCF secretion from monocytes as effectively as untreated ones. On the other hand, actinomycin D treatment of K562 cells enhanced their susceptibility to NKCF and MCF. This actinomycin-D-induced augmentation of target sensitivity to the cytotoxic factors was restricted to NK-sensitive target cells (K562 and Molt-4). NK-resistant target cells (Raji, YAC-I, EL4 and T blasts) were not lysed by NKCF and MCF even after they were treated with actinomycin D. The capacity of K562 cells to bind NKCF and MCF was not altered by actinomycin D. Treatment of the adherent cell population with OKMI or Leu-MI plus complement abrogated both cell-mediated cytotoxicity and MCF production, while Leu-IIb plus complement was ineffective. These results suggest that the effect of actinomycin-D treatment can be used to distinguish the two distinct types of blood mononuclear cells with natural cytotoxicity, NK cells and monocytes, and that each effector type recognizes different plasma membrane moieties of NK target cells, although the cytotoxic factors released from each effector cell similarly bind to and lyse the target cells.     

Cytotoxicity responses to melanoma cells by human lymphoid cell subpopulations.

Lymphoid cell subpopulations from normal donors and patients with malignant melanoma were assessed for cytotoxicity. Unfractionated mononuclear cells and T-cells from melanoma patients gave cell-mediated cytotoxic (CMC) responses to melanoma target cells but not to human fibroblasts. These specific CMC responses to melanoma cells were partially inhibited by autologous serum. Non-T-cells and nonrosetting cells from melanoma patients were not directly cytolytic for melanoma target cells; however these same subpopulations were cytotoxic in the presence of autologous serum, which indicated antibody-dependent cell cytotoxic responses. Non-T-cell subpopulations from normal donors were not cytotoxic when incubated with autologous serum. A third cytotoxic mechanism was demonstrated with complement (C3) receptor-activated lymphocytes. From both melanoma patients and normal donors, cells forming rosettes with erythrocyte-antibody-complement were nonspecifically cytotoxic for lung fibroblasts and melanoma target cells, These responses were independent of antibody, since melanoma serum presumably containing antibody to melanoma target cells neither enhanced nor blocked cytotoxicity mediated by C3 receptor-bearing lymphocytes. The results indicated that lymphoid cell subpopulations from the same melanoma patient could express at least three different lymphocyte-mediated cytotoxicity mechanisms against melanoma target cells.     

The influence of different isolation procedures and the use of target cells from melanoma cell lines and short-term cultures on the non-specific cytotoxic effects of lymphocytes from healthy donors

In parallel studies the effects of FHL¹ and PNL on plated melanoma cells from cell lines and short-term cultures were compared. FHL showed more frequent and also stronger cytotoxic and/or growth-inhibiting effects than PNL. On melanoma target cells from cell lines both FHL and PNL showed more frequent and stronger cytotoxic and/or growth-inhibiting effects than on melanoma target cells from short-term cultures. In the individual donors the percentage of monocytes and EAC-rosette-forming cells in FHL was significantly higher than in PNL. A significant correlation was found between multiplication of the melanoma target cells during the test period and an increased susceptibility towards lymphocytes from healthy donors. Melanoma target cells from cell lines were not more fragile, or more susceptible to unfavourable culture conditions than cells from short-term cultures, since non-lymphocytic ?effector”? cells showed much weaker cytotoxic and/or growth-inhibiting effects than lymphocytes from healthy donors. Cytotoxic effects of lymphocytes from healthy donors were also registered on target cells from a mammary carcinoma and an osteosarcoma cell line. No significant differences in the cytotoxic effects of lymphocytes from healthy donors were observed when tested on mycoplasma-contaminated melanoma cells and the same cells made mycoplasma-free. Mitomycin-C-treated lymphocytes retained their cytotoxic effects. Lymphocytes from a healthy donor tested on different occasions on the same melanoma cells from a short term culture showed an incidental cytotoxic reaction.     

Natural cytotoxicity of human blood lymphocytes and monocytes and their cytotoxic factors: effect of interferon on target cell susceptibility

The effect of interferon (IFN) on target cell susceptibility to human natural killer (NK) cells and monocytes was analyzed in direct cell-mediated and their cytotoxic factor-mediated cytotoxicity assays. Treatment of K562 cells with IFN resulted in a decrease in their sensitivity to lysis by nonadherent lymphocytes and Percoll-purified large granular lymphocytes (LGL) when tested in a 4-hour 51Cr release assay. In contrast, the treatment did not affect the target susceptibility to monocytes purified by adherence to autologous serum-coated plastic surfaces. In the target-binding assay with LGL or monocytes the number of conjugates was not altered after IFN treatment of K562. Lymphocytes and monocytes were induced to release soluble cytotoxic factors, termed "natural killer cytotoxic factors (NKCF) and monocyte cytotoxic factors (MCF)," respectively, when co-cultured with K562. Both NKCF and MCF lysed K562 in a 48-hour microcytotoxicity assay or in an 18-hour 51Cr release assay in the presence of dactinomycin. IFN-treated K562 reduced or completely lost their ability to stimulate the release of NKCF, whereas they triggered MCF secretion as effectively as did the untreated K562. When lymphocytes or monocytes were pretreated with IFN, they released NKCF or MCF with augmented lytic activity. In contrast to the sensitivity to NK cell-mediated lysis, IFN pretreatment of K562 induced no change in their susceptibility to NKCF and MCF. When IFN was added to NKCF and MCF assays, the cytotoxicity was enhanced. The addition of IFN to K562 that had been pretreated with NKCF or MCF and washed resulted in no increase in lysis. The capacity of K562 to absorb the lytic activity of NKCF and MCF was not altered by IFN. These results indicate that IFN treatment of target cells can be used to distinguish the two distinct types of blood mononuclear cells with natural cytotoxicity, NK cells and monocytes, and that each effector cell type is stimulated to release cytotoxic factors by the different target determinant after the initial effector-target cell binding.     

Antibody dependent cell mediated cytotoxicity on human cervical carcinoma cell line, ME-180, with human monoclonal antibody.

A human monoclonal antibody (MCA), CLN-IgG, showed cytotoxic effect in vitro against the cervical carcinoma cell line, ME-180, by antibody dependent cell-mediated cytotoxicity (ADCC). To determine which fractions of cells in peripheral blood lymphocyte (PBL) mediate ADCC, PBL were separated with nylon wool column and sheep red blood cells (SRBC). Both adherent cells (monocyte) and non-T, non-B cells showed cytotoxicity by ADCC. Human non-T, non-B cells showed higher cytotoxic activity against ME-180 cells than monocytes. Furthermore murine effector cells were less effective in ADCC than human effector cells with human MCA.     

Activation of cytolytic activity in peripheral blood

Our purpose was to evaluate the ability of blood monocytes of renal cancer patients to become cytotoxic against fresh, autologous tumor cells. Fresh target cells were obtained by mechanical and enzymatic dissociation of tumor and normal renal tissue. The A375 cell line, derived from a human melanoma, and the SW626 cell line, derived from a human ovarian carcinoma, were used as positive target cell controls. Monocytes from renal cancer patients and normal volunteers were activated in vitro with lipopolysaccharide (LPS), or muramyl tripeptide (MTP-PE), or multilamellar vesicle liposomes containing MTP-PE (MLV-MTP-PE), with or without a pre-incubation with r-IFN-γ, and tested for cytotoxicity in a 72-hr ¹¹¹Indium-release assay. All patients were tumor-free at the time of the monocyte study. No difference in cytotoxic activity was observed between monocytes from healthy volunteers and those from cancer patients. Freshly dissociated tumor cells were as susceptible to tumoricidal monocytes as the 2 cell lines. Moreover, no cell population appeared to be resistant to activated monocytes, which were cytotoxic to both allogeneic and autologous fresh tumor cells. Activated monocytes maintained their ability to discriminate between normal and neoplastic cells and were not cytotoxic against autologous or allogeneic normal non-neoplastic cells. Our data indicate that MLV MTP-PE liposomes activate peripheral blood monocytes from cancer patients to a tumoricidal status against fresh, dissociated non-cultured autologous tumor cells.     

Disease-related lymphocyte cytotoxicity in rats bearing transitional cell carcinoma of bladder. effect of immunomodulators

Blood lymphocytes from rats bearing transitional cell carcinoma (TCC) of the bladder were studied for their cytotoxicity in vitro against xenogeneic YAC-1 target and against syngeneic TCC cells. Control lymphocytes were obtained from age and sex-matched syngeneic rats. The following differences were observed: (I) lymphocytes from TCC-bearing rats were cytotoxic to syngeneic TCC target cells whereas those from control rats were not; (2) lymphocytes from TCC-bearing and control rats were cytotoxic to NK sensitive YAC-I cells; however, NK cells from TCC-bearing rats were more adherent to nylon wool-columns than NK cells from control rats. The adherent and non-adherent cells from TCC-bearing rats were both cytotoxic to syngeneic TCC target cells. Levamisole treatment of effector cells from TCC-bearing rats did not affect the NK activity, yet it increased the cytotoxicity of non-adherent cells on TCC target cells. Treatment of the adherent cells with poly-l:poly-C increased slightly their NK activity on YAC-I cells and their anti-TCC cytotoxicity. However, a marked increase in the cytotoxicity by both adherent and non-adherent cell fractions was observed on TCC target cells pretreated with poly-l:poly-C. A disease-related cytotoxicity of lymphocytes from rats bearing TCC has been observed. Treatment of TCC target cells with poly-l:poly-C increased their susceptibility to lysis by the activated effector cells.     

Bacillus Calmette-Guerin (BCG) enhances monocyte- and lymphocyte-mediated bladder tumour cell killing.

A cytotoxicity assay was used to study the action of bacillus Calmette-Guerin (BCG) and cytokines on four human bladder cancer cell lines. Monocytes and lymphocytes from peripheral blood were incubated with or without BCG or cytokines for 24 h, after which [3H]thymidine-labelled target cells were added and the 72 h percentage specific release determined. BCG had a direct cytotoxic effect against tumour cells and significantly enhanced monocyte/macrophage and enhanced lymphocyte cytotoxicity against one cell line (UCRU-BL-17). Supernatants (SNs) from BCG-activated monocytes/macrophages and lymphocytes increased the percentage specific release of [3H]thymidine from UCRU-BL-17 cells. Interferon alpha (IFN-alpha) and interleukin 2 (IL-2) were cytotoxic towards UCRU-BL-17. No synergy occurred between BCG and cytokines at the concentrations tested. The results suggest that BCG is superior to IFN-alpha, interferon gamma (IFN-gamma) and IL-2 in enhancing cell-mediated cytotoxicity.     

Tumor necrosis factor as effector molecule in monocyte mediated cytotoxicity

A newly developed assay system which uses actinomycin D (Act D) pretreated Wehi 164 target cells allows for the measurement of human monocyte cytotoxicity in a 7-h 51Cr release assay. Using the monocyte specific monoclonal antibody M42 in a direct rosetting procedure we confirm herein that among human peripheral blood mononuclear cells cytotoxicity is restricted to monocytes. When applying stringent conditions that exclude exogenous lipopolysaccharide (LPS) we could demonstrate that as little as 0.1 ng of LPS per ml triggers this cytotoxicity. Further, a factor can be detected in supernatants of mononuclear cells which is also cytotoxic against Act D treated Wehi 164 cells. This cytotoxic factor can be triggered by LPS within 4 h, but at as low a LPS concentration as 0.001 ng/ml. Since one of the LPS triggered monocyte products is tumor necrosis factor (TNF), we tested the effect of recombinant TNF cloned from the U937 cell line and we could show potent lytic activity against Act D pretreated but not, or only minimally, against untreated Wehi 164 target cells. Recombinant TNF rapidly lysed the target with significant specific release occurring as early as after 3 h in the assay. By contrast, recombinant interleukin 1 gave no lysis while lymphotoxin derived from the RPMI 1788 cell line was effective. An affinity purified antiserum directed against TNF neutralized the lytic activity of recombinant TNF and also the cytotoxic factor produced by LPS triggered mononuclear cells, while the antiserum was ineffective against lymphotoxin. Further, the antiserum when added to the assay of effector cells and Act D treated Wehi 164 cells also completely ablated cytotoxic activity. Size fractionation of cytotoxic factor and recombinant TNF by high pressure liquid chromatography led to a superimposable peak of cytotoxicity in the molecular weight range of 9,500-17,000. Further, immunoblotting with the anti-TNF antibody revealed the same Mr 15,500-16,500 band for the recombinant TNF and LPS triggered cytotoxic factor. Taken together, our data demonstrate that the cytotoxic activity of human monocytes against Act D treated Wehi 164 is mediated entirely by a LPS triggered molecule that is very similar or identical to the human tumor necrosis factor. The assay system thus provides a powerful tool to analyze the biology of TNF in humans.     

Susceptibility of NB-I Neuroblastoma Cells to Tumoricidal Activity of Monocytes Activated by γ-Interferon

The purpose of this study was to examine the susceptibility of NB-I human neuroblastoma cells to direct cellular cytotoxicity mediated by peripheral blood monocytes from pediatric cancer patients receiving chemotherapy. Nonactivated monocytes from patients showed spontaneous cytotoxicity to NB-I neuroblastoma cells (37 ± 18%) but only marginal cytotoxicity to A375 melanoma cells (21 ± 14%) at the effector:target cell ratio of 20:1. This spontaneous cytotoxicity to NB-I cells was observed only after >24 h of cocultivation and was proportional to the effector:target cell ratio. Activation of monocytes by recombinant human interferon γ (rIFN) (1×10⁴ U/ml) consistently and strongly enhanced their tumoricidal activity to NB-I cells (87 ± 6%) and this tumoricidal activity was even superior to that observed against A375 cells, which are known to be extremely sensitive to lysis by activated monocytes. In contrast, activation of monocytes by lipopolysaccharide (LPS, 1 μg/ml) had no effect on monocyte-mediated lysis of NB-I cells, while A375 cells were equally lysed by rIFN- and LPS-activated monocytes, thus suggesting that different mechanisms are involved in the monocyte-mediated lysis of A375 melanoma and NB-I neuroblastoma cells. Susceptibility of the neuroblastoma cell line to monocyte-mediated cytotoxicity has not been reported so far and our results may have some clinical implication if this observation can be extended to other neuroblastoma cell lines as well.     

Susceptibility of NB-I neuroblastoma cells to tumoricidal activity of monocytes activated by gamma-interferon

The purpose of this study was to examine the susceptibility of NB-I human neuroblastoma cells to direct cellular cytotoxicity mediated by peripheral blood monocytes from pediatric cancer patients receiving chemotherapy. Nonactivated monocytes from patients showed spontaneous cytotoxicity to NB-I neuroblastoma cells (37 +/- 18%) but only marginal cytotoxicity to A375 melanoma cells (21 +/- 14%) at the effector:target cell ratio of 20:1. This spontaneous cytotoxicity to NB-I cells was observed only after greater than 24 h of cocultivation and was proportional to the effector:target cell ratio. Activation of monocytes by recombinant human interferon gamma (rIFN) (1 x 10(4) U/ml) consistently and strongly enhanced their tumoricidal activity to NB-I cells (87 +/- 6%) and this tumoricidal activity was even superior to that observed against A375 cells, which are known to be extremely sensitive to lysis by activated monocytes. In contrast, activation of monocytes by lipopolysaccharide (LPS, 1 microgram/ml) had no effect on monocyte-mediated lysis of NB-I cells, while A375 cells were equally lysed by rIFN- and LPS-activated monocytes, thus suggesting that different mechanisms are involved in the monocyte-mediated lysis of A375 melanoma and NB-I neuroblastoma cells. Susceptibility of the neuroblastoma cell line to monocyte-mediated cytotoxicity has not been reported so far and our results may have some clinical implication if this observation can be extended to other neuroblastoma cell lines as well.     

Chimeric and humanized anti-HM1.24 antibodies mediate antibody-dependent cellular cytotoxicity against lung cancer cells

HM1.24 antigen (CD317) was originally identified as a cell surface protein that is preferentially overexpressed on multiple myeloma cells. Immunotherapy using anti-HM1.24 antibody has been performed in patients with multiple myeloma as a phase I study. The aim of this study was to evaluate the anti-tumor activity of mouse-human chimeric and humanized anti-HM1.24 monoclonal antibodies (mAbs) against lung cancer cells in vitro. Human peripheral blood lymphocytes and monocytes separated from mononuclear cells (PBMCs) were used as effector cells. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of chimeric and humanized anti-HM1.24 mAbs against lung cancer cells were determined by chromium-release assay. In some experiments, target or effector cells were pretreated with various cytokines. Chimeric and humanized anti-HM1.24 mAbs effectively induced ADCC against lung cancer cells mediated more efficiently by lymphocytes than monocytes. The cytotoxic activity correlated with the level of HM1.24 expression on lung cancer cells. Natural killer cells were identified as the major effector cells in ADCC mediated by the anti-HM1.24 mAb. The treatment of lymphocytes or monocytes with IL-2, IL-12, IL-15, M-CSF, or IFN-gamma significantly increased the ADCC activity. Moreover, the culture of lung cancer cells with IFN-beta or IFN-gamma augmented their susceptibility to ADCC and CDC. PBMCs from patients with lung cancer induced a level of ADCC comparable to that induced by PBMCs from healthy donors. Chimeric or humanized anti-HM1.24 mAbs have potential as a new therapeutic tool in lung cancer, and in combination with interleukins and interferons, could be useful for enhancing ADCC.     

Role of monocyte cytotoxic factor in cytolysis of actinomycin D-treated WEHI 164 cells mediated by freshly isolated human adherent mononuclear blood cells

The role of the monocyte cytotoxic factor (CF) in cytolysis of untreated and actinomycin D (Act D)-treated WEHI 164 cells by freshly isolated human adherent mononuclear cells has been investigated in this study. Murine WEHI 164 cells were used as target cells because of their sensitivity to lysis mediated by monocytes and their resistance to natural killer cells. Monocytes as well as monocyte supernatants mediated cytolysis of WEHI 164 cells. Cytolysis was enhanced by Act D treatment of target cells. The addition of lipopolysaccharide to monocytes accelerated the progression of cytolysis of Act D-treated WEHI 164 cells mediated by monocytes. A polyclonal rabbit antiserum against CF inhibited the cytolytic activity of monocytes and monocyte supernatants against untreated as well as Act D-treated WEHI 164 cells. At low effector:target ratios, the cytolysis was totally abrogated by CF antiserum. Depletion of natural killer cells from adherent cells by the monoclonal antibody Leu 11b and rabbit complement did not reduce cytolysis of Act D-treated WEHI 164 cells. Immunofluorescence microscopy revealed that CF antiserum stained the plasma membrane of freshly isolated monocytes, suggesting that CF is a membrane-associated molecule. Our data indicate that CF is an important effector molecule in cytolysis mediated by freshly isolated monocytes against untreated and Act D-treated WEHI 164 cells.     

Effects of HC antibody in autologous tumor-specific cytotoxicity by human melanoma tumor-infiltrating lymphocytes

Heteroconjugate (HC) antibody has a potential use in cancer biotherapy because of its ability to mimic antigenic specificity and induce cytotoxicity in the activated lymphocytes against various tumor cells. This study investigated the effects of HC antibody (anti-CD3 MAb x anti-p97 melanoma cell MAb) in autologous tumor-specific cytotoxicity by interleukin-2 (IL-2)-activated melanoma tumor-infiltrating lymphocytes (TILs). HC antibody significantly augmented p97pos uncultured autologous tumor cell lysis mediated by effector TILs or cytotoxic T lymphocyte (CTL) clones derived from TIL. It did not significantly increase p97mix autologous tumor-cell lysis and slightly inhibited the lysis only at higher E:T ratios and higher concentrations (greater than or equal to 100 ng/ml). It inhibited p97neg autologous tumor-cell lysis. HC antibody respectively induced potent lysis of p97pos or modest lysis of p97mix tumor cells by allogeneic effector TILs as well as PBMC. In contrast, parental anti-CD3 MAb primarily suppressed the autologous tumor-specific cytotoxicity, and did not induce lysis of uncultured melanoma cells, regardless of differences in expression of p97 antigens on tumor cells. Although parental anti-p97 MAb did not augment or suppress the autologous tumor-specific cytotoxicity, it completely abrogated HC antibody-mediated augmentation of p97pos autologous tumor cell lysis by effector TILs. Anti-class-I MAb, but not anti-DR MAb, suppressed the autologous tumor-specific cytotoxicity, but failed to block HC antibody-mediated augmentation of p97pos autologous tumor-cell lysis. These results suggest that the levels of p97 antigen expression largely influenced HC antibody-mediated modulation of TIL cytotoxicity against uncultured autologous tumor cells.     

Cytolytic and cytostatic activity on tumor cells of circulating human monocytes.

Monocytes from the peripheral blood of normal adult human donors were found to have appreciable levels of cytotoxic activity against murine and human tumor-cell lines. Adherent cells ( > 90% monocytes) were obtained from the peripheral blood of 29 normal healthy volunteers either by adherence on plastic and scraping with a rubber policeman or by adherence on microexudate-coated plastic and exposure to ethylene diamine tetra-acetic acid. Cytolytic capacity was tested by incubating effector cells for 72 h with murine and human tumor cell lines prelabelled with tritiated thy-midine. Cytostasis was evaluated by inhibition of [¹²⁵I]iododeoxyuridine (¹²⁵IdUrd) uptake. Tumor target cells employed were: a murine SV40-trans-formed kidney line (TU5), a murine chemically-induced sarcoma (1023), a human breast-cancer-derived cell line (G11) and a human lung-cancer-derived cell line (CaLu). Monocyte preparations at attacker to target cell ratios of 1:1 to 40:1, showed significant cytolytic and cytostatic activity against tumor target cells. Tumor cells showed different susceptibility to cytolytic activity, whereas comparable levels of cytostasls were observed with the various targets: TU5 and G11 tumor cells were more susceptible than 1023 and CaLu target cells to the cytolytic capacity of human monocytes and TU5 was used in most subsequent experiments. Peak isotope release from prelabelled target cells was observed after 72 h of incubation, whereas peak inhibition of [¹²⁵I]dUrd uptake occurred after 24 h of culture. The cytotoxic capacity of monocytes isolated by either of the two methods mentioned above was similar. The monocytes had higher cytotoxic activity than unseparated mononuclear cells, and non-adherent cells showed minimal cytotoxic effects. Cytotoxicity by natural killer cells did not appear to have a major role in these assays, since adherent cells did not lyse K562 cells in a 4-h ⁵¹Cr release assay. Treatment with anti-human T-cell serum and complement did not inhibit the cytotoxic capacity of the monocyte preparations, whereas exposure to silica particles significantly inhibited the cytotoxic activity. Monocyte-mediated cytotoxicity on tumor cells was expressed in the presence of both fetal bovine serum and human AB serum.